Compositions and Methods for Identifying and Treating Dystonia Disorders

ABSTRACT

The present disclosure provides methods and compositions for the treatment, identification, diagnosis, and prognosis of dystonia, or dystonia related disorders.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the diagnosis, prognosis, and treatmentof dystonia and dystonia related disorders.

Description of Related Art

Dystonia is a movement disorder characterized by sustained, oftenpainful involuntary postures, causing patients motor disability and amarked decrease in quality-of-life. As a class of disorders, dystonia isthe 3rd most common movement disorder behind Parkinson and essentialtremor, and is present as a symptom in a broad range of clinicalcontexts (e.g. sporadic, neurodegeneration, trauma, medication sideeffects). Once symptoms onset, they are typically unremitting, creatinga substantial quality of life impairment and disease burden.Importantly, the therapeutic armamentarium for dystonia is severelylimited.

DYT1 early-onset torsion dystonia is a severe, childhood-onset form ofdystonia. DYT1 dystonia is caused by an in-frame trinucleotide deletionin the TOR1A gene, leading to the loss of a single glutamic acid residue(delta-E) from the AAA+ATPase Torsin1a. (See, e.g., Ozelius et al.,1997, Nat. Genet. 17, 40-8.) Though the precise function of Torsin1a isunknown, the delta-E mutation has a dramatic effect in its subcellularlocalization. (See, e.g., Goodchild, et al. 2005, J. Cell Biol. 168,855-62; Kustedjo et al. 2000, J. Biol. Chem. 275, 27933-9; Bragg et al.2004, Neuroscience 125, 651-61; Calakos et al. 2010, J. Med. Genet. 47,646-50; Gonzalez-Alegre et al. 2004, J. Neurosci. 24, 2593-601;Goodchild et al. 2004, Proc. Natl. Acad. Sci. U.S.A 101, 847-52; Hewettet al., 2008, Hum. Mol. Genet. 17, 1436-45; Kock et al. 2006, Hum. MoLGenet. 15, 1355-64; Hewett et al. 2000, Hum. Mol. Genet. 9, 1403-13;Vander Heyden et al. 2009, Mol. Biol. Cell 20, 2661-72; Goodchild et al.2005, J. Cell Biol. 168, 855-62; Martin et al. 2009, Neuroscience 164,563-72; Liang et al. 2014, J. Clin. Invest. 124, 3080-92; Giles et al.2008, Hum. Mol. Genet. 17, 2712-22; Vulinovic et al. 2014, Hum. Mutat.35, 1114-22.)

Normally, the wildtype (WT) Torsin1a cycles between the outer nuclearenvelope (NE) and the lumen of the endoplasmic reticulum (ER) in an ATPhydrolysis-dependent fashion, with the bulk of the protein detected inthe ER. See, e.g., Goodchild et al. 2004; Naismith et al. 2004, Proc.Natl. Acad. Sci. U.S.A 101, 7612-7. In contrast, when delta-E Torsin1ais the major species, as in overexpression experiments or homozygousknock-in mouse models, it predominantly co-localizes with nuclearenvelope (“NE”) markers and disrupts the normal subcellular NE membranestructure in a manner that is suggestive of a membrane-traffickingdefect. (Goodchild et al, 2004; Naismith et al. 2004; Jokhi et al, 2013,Cell Rep. 3, 988-95.)

At the light microscopic level, delta-E Torsin1a distribution appears asan abnormal punctate pattern (see FIG. 1, panel a); an appearance thatis in striking contrast to the diffuse reticular pattern of similarlyexpressed WT Torsin1a (see FIG. 1b ). Because delta-E Torsin1amis-localization is a robust, early phenotype associated with disruptionof basic cellular architecture, it is possible that molecular pathwaysremediating delta-E Torsin1a mis-localization could serve as noveltherapeutic targets for Dyt dystonia.

The etiology of other forms of inherited and sporadic dystonia are lessclear or entirely unknown. Furthermore, current drug treatments are onlysymptomatic, of modest efficacy, and are poorly tolerated because ofside effects (anticholinergics). Other treatment options are invasiveand generally require access to tertiary care centers (deep brainstimulation, botulinum toxin).

Thus, there is a need in the art for methods of diagnosing and treatingsubjects suffering from inherited and sporadic forms of dystonia,including understanding the molecular basis of the disease. Furthermore,there is a need in the art to understand the basis for aberrant delta-ETorsin1a localization in DYT1 dystonia patients.

SUMMARY OF THE INVENTION

Against this backdrop, embodiments of the present disclosure address oneor more of the above-identified needs, among others, recognized by thoseskilled in the art, and provide several benefits over existing clinicalmethods of identification, diagnosis, prognosis, and therapeuticintervention in, or treatment of, subjects with dystonia.

In embodiments, the disclosure provides methods of treating dystoniausing an HIV aspartyl protease inhibitor. Examples include, but are notlimited to, ritonavir, liponavir, saquinavir, nelfinavir, and indinavir.In some embodiments, an inhibitor of the disclosure is selected fromritonavir, liponavir, saquinavir, and combination thereof.

In some embodiments, the inhibitor of the disclosure is a compound offormula (I):

-   or pharmaceutically acceptable salts thereof, wherein-   each n is independently selected from 0, 1, 2, 3, and 4;-   R¹ is hydrogen or C₁-C₆ alkyl;-   R² and R³ are each independently selected from halogen, hydroxy,    C₁-C₆ alkyl, and C₁-C₆ alkoxy; and-   R⁴ is —C(O)R⁸, —C(O)OR⁸, —C(O)NHR⁸, or —C(O)N(C₁-C₆ alkyl)R⁸,    -   where R⁸ is C₁-C₆ alkyl, aryl, heteroaryl, heterocyclyl,        arylC₁-C₆ alkyl-, heteroarylC₁-C₆ alkyl-, or heterocyclylC₁-C₆        alkyl-, each optionally substituted with one or more of groups        selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆        alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino;-   R⁵ is selected from hydrogen and C₁-C₆ alkyl,-   or R⁵ and R⁸ together with atoms to which they are attached form    heterocyclyl optionally substituted with one or more of groups    selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆    alkoxy, amino, C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo, or thio;    and-   R⁶ is —Z—R⁷, wherein    -   Z is absent, —C₁-C₆ alkylene-, —O—C₁-C₆ alkylene-, —C₁-C₆        alkylene-O—, —NH—C₁-C₆ alkylene-, —C₁-C₆ alkylene-NH—, —O—,        —NH—, or —N(C₁-C₆ alkyl)-; and    -   R⁷ is aryl, heteroaryl, or heterocyclyl, each optionally        substituted with one or more of groups selected from halogen,        cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆        alkylamino, and diC₁-C₆ alkylamino.

In other embodiments, compound of formula (I) is of formula (I-A):

In further embodiments, the inhibitor of the disclosure is a compound offormula

-   or pharmaceutically acceptable salts thereof, wherein-   m is selected from 0, 1, 2, 3, and 4;-   q is selected from 0, 1, 2, 3, and 4;-   X is —O—, —NH—, —CHR¹⁴—, —C(R⁴)₂—, or —CH₂—,-   R¹¹ is aryl, heteroaryl, or heterocyclyl, each optionally    substituted with one or more of groups selected from halogen, cyano,    nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino,    and diC₁-C₆ alkylamino;-   R¹² is —C(O)R¹⁵, —C(O)OR¹⁵, —C(O)NHR¹⁵, or —C(O)N(C₁-C₆ alkyl)R¹⁵,    -   where R¹⁵ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl,        heteroaryl, heterocyclyl, arylC₁-C₆ alkyl-, heteroarylC₁-C₆        alkyl-, or heterocyclylC₁-C₆ alkyl-, each optionally substituted        with one or more of groups selected from halogen, cyano, nitro,        C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and        diC₁-C₆ alkylamino;-   R¹³ is each independently selected from halogen, hydroxy, C₁-C₆    alkyl, and C₁-C₆ alkoxy; and-   R¹⁴ is each independently selected from halogen, cyano, nitro, C₁-C₆    alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆    alkylamino;-   or two R¹⁴ with atoms to which they are attached form heterocyclyl    optionally substituted with one or more of groups selected from    halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino,    C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo, or thio.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and other features of the invention are explainedin the following description, taken in connection with the accompanyingdrawings, in which:

FIG. 1 shows results of delta-E (“AE”) Torsin1a mis-localization assayand whole genome siRNA screen. Panels (a) and (b) show representativeimages of Flp-In T-REx 293T stable cell lines expressing delta-E (panela) and wild-type (“WT”) (panel b) EGFP-hTorsin1a following 72 htetracycline induction (light punctate staining comprises EGFP-hTorsin1asignal; diffuse darker staining comprises Hoescht nuclear stain). Scalebars=10 μm. Panel (c) shows that silencing Torsin1b (TOR1B) worsensTorsin1a mis-localization in both WT and delta-E cell lines. Panel (d)shows that proteasomal inhibition with MG132 increases Torsin1amis-localization, and the chemical “chaperone,” phenylbutyric acid (PBA)reduces Torsin1a mis-localization. Panels (e)-(g) show representativelow-magnification images of cell lines acquired under high-throughputscreen conditions in 384-well plates following reverse transfection withcontrol siRNAs. WT (panel e) and delta-E treated with non-silencingsiRNA control (panel f) or with positive control siRNA (panel g). Scalebars=50 μm. Panel (h) shows assay reproducibility and siRNA poolingstrategy. Inset—Four independent siRNAs targeting each gene were splitinto two unique pools of two siRNAs. Panel (1) shows results of a wholegenome siRNA (WGS) screen. Dots represent data for individual genetargets, with results from each independent siRNA pool plotted onorthogonal axes. Panel (j) shows a schematic depicting WGS workflow foranalyzing primary hits. Panel (k) shows performance of four of top 93hits in counter screen to rescue deficient secretion in fibroblastsderived from human DYT1 patients relative to WT cells. All data arepresented as means t S.E.M. * denotes p<0.05; *** denotes p<0.0005 byunpaired t test. Panel (I) shows strategy for site-specific integrationof a single cDNA copy of EGFP-WT-Torsin1a or EGFP-deltaE-Torsin1a intoFlp-In HEK 293 T-REx cells under control of a tetracycline-sensitivetranscriptional repressor.

FIG. 2 shows that enhancement of eIF2-alpha (also referred to as“eIF2α”) corrects delta-E Torsin1a mis-localization. Panel (a) shows adiagram of the eIF2-alpha signalling pathway. Proteins whose knockdownworsened the percentage of cells with punctate delta-E Torsin1a in thewhole genome screen (WOS) are indicated by circles. Panel (b) shows WGSresults relevant to the eIF2-alpha pathway. Dashed lines indicate 3Standard Deviations from mean (solid line). Panels (c-h), left panels,show the effects of the indicated compounds on Torsin1a localization(black circles), cell count (grey squares), and EGFP-Torsin1a expression(grey triangles) in the delta-E (panels c-e, g, h) or WT (panel f) assaycell lines. Right panels show representative images from the respectivetreatments. Scale bars=20 μm. All data are presented as means±S.E.M.Panels (I) and (j) show that DMSO vehicle control has no effect on WTTorsin1a localization (i) or cell count (j).

FIG. 3 shows that ATF4 overexpression is sufficient to correct delta-ETorsin1a mis-localization. Panel (a) shows representative images ofEGFP-Torsin1a localization (light punctate staining) and FLAG epitopestaining (white arrows) in the transfected delta-E and WT assay celllines. Scale bars=20 μm. Panel (b) shows quantitation of data presentedin panel (a). All data are presented as means±S.E.M. ***, p<0.0005 byunpaired t test.

FIG. 4 shows that salubrinal improves perinatal survival of homozygousDYT1 knock-in mice. Panel (a) shows the experimental design of perinatalsurvival experiments. Panel (b) shows the effect of in utero salubrinalexposure on perinatal survival. **, p<0.01 by Chi-square test.

FIG. 5 shows molecular and genetic analyses establishing eIF2α pathwaydysfunction in DYT1 and sporadic dystonia patients. Panel (a) showsWestern blot analysis of ATF4 levels in fibroblasts derived from DYT1patients, and healthy control cells, at various times followingthapsigargin treatment. Blots were re-probed for β-actin as a loadingcontrol. Panel (b) shows quantitation of the data presented in panel(a). **, p<0.005 by two-way ANOVA. Panel (c) shows the results of wholeexome sequencing of patients with sporadic dystonia patients and normalcontrols. Among these is a p.P46L mutation in ATF4, an eIF2-alphapathway gene. Panel (d) shows that the P46L mutation reduces ATF4transcriptional activity relative to WT or another common ATF4 variant(Q22P). **, p<0.005 by unpaired t test. Panel (e) shows representativeWestern images revealing reduced steady-state levels of P46L ATF4relative to WT controls. ATF4 expression was normalized to GAPDH. Panel(f) shows quantification of reduced P46L FLAG-ATF4 levels. *, p<0.05 byunpaired t test. Panel (g) shows the ratio of ATF4 levels (normalized toGAPDH) under proteasomal inhibition relative to untreated(MG132/untreated) controls. **, p<0.005 by unpaired t test. All data arepresented as means±S.E.M.

FIG. 6 shows additional sporadic mutations in dystonia patients withreduced ATF4 transcriptional activity relative to WT controls. Panel (a)shows transcriptional activation activity of mutant ATF4 constructsmeasured in HEK293T cells. Data was normalized such that luciferaseactivity after WT ATF4 transfection=1. *, p<0.05; ***, p<0.0005 byunpaired t test vs WT ATF4 condition. Panel (b) shows rare and commonvariants in ATF4 exon 1 identified by Sanger sequencing of 239 sporadiccervical dystonia patients; frequency, enrichment and predicted mutationseverity shown at right.

FIG. 7 shows that the eIF2-alpha phosphatase CreP is constitutivelyupregulated in DYT1 dystonia patients, and is not further upregulated inresponse to thapsigargin. Panel (a) shows representative Western blotsfor CreP with and without exposure to thapsigargin. Blots were re-probedfor 3-actin as a loading control. Panel (b) shows quantitation ofreplicates of the Western analyses presented in panel (a). N=3independent control cell lines, and 4 independent DYT1 cell lines, 3replicates each. *, p<0.05 by unpaired t test. All data are presented asmeans±S.E.M. CreP expression was normalized to β-actin expression. Panel(c) shows quantitation of the fold-change in CreP levels in control andDYT1 fibroblasts upon thapsigargin exposure (1 μM, 4 h). Panel (d) showsa model depicting the sites of eIF2-alpha pathway dysfunction in variousforms of dystonia.

FIG. 8 shows restoration of long term plasticity in the cortico-striatalsynapse by modulation of eIF2-alpha pathway. Panel (a) shows a timecourse of Long term depression (LTD) in cortico-striatal synapse inducedby 4 trains 100 Hz on V layer of cortex (see Example 10) in WT mice(vehicle; white dots), and when incubated with ISRIB (5 nM, 1 hr.)(black dots; P=0.015). Panel (h) shows the temporal course indelta-E-TorsinA mice treated with vehicle; (white dots), or sa1003 (20mM; shaded dots; p=0.0274). In each of panels (a) and (b), black tracesabove the graph are EPSPs recorded before HFS, gray traces are EPSPsrecorded after 21 min post HFS. Panel (c) shows mean magnitude of LTD in(A) and (B). Data in blue shaded box in panels (A) and (B) (minutes21-31) were averaged. *, p<0.05 by Mann-Whitney U-test. Data aremeans±S.E.M.

FIG. 9 shows high-content image analysis and assay performance underhigh-throughput screening conditions. Panels (a) and (b) show automatedimage analysis and determination of EGFP-Torsin1a localization usingCellomics compartmentalization protocol software. Assay output (definedas % selected cells) is the percentage of cells that are identified withpuncta. Panel (c) shows the time course of the high-throughput,high-content RNAi screening assay. Panel (d) shows that the percentageof selected cells was robustly higher in the AE compared to WT Torsin1aassay cells (Z′=0.614 t 0.077), and treatment with a Positive Control(PC) siRNA reliably led to a marked reduction in the percent of selectedcells. n=96 independent wells per condition. ***, p<0.0005 by unpaired ttest. Data in (d) are presented as box-and-whisker plot displaying 90%confidence interval. Panel (e) shows that TOR1A siRNA mediated silencingof assay readout protein (EGFP-Torsin1a) reduces the percent of selectedcells to near zero. n=16 independent wells treated with non-silencingsiRNA control and 32 independent wells treated with TOR1A siRNA. Data in(e) are presented as means±S.E.M.

FIG. 10 shows Torsin1a mis-localization is restored in cells treatedwith ritonavir.

FIG. 11 shows that lopinavir treatment in EGFP-delta-E Torsin1a Flip-InTREx 293 cells decreases CReP abundance. Panel (a) shows Western blotanalysis of CreP and eIF2-alpha in WT and delta-E Torsin1a cells exposedto increasing concentration of Lopinavir. Blots were probed for β-actinas a loading control. Panel (b) shows quantitation of CreP proteinlevels, and panel (c) shows quantitation of eIF2-alpha protein levels,in WT and delta-E Torsin1a cells exposed to varying doses of lopinavir.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alteration and furthermodifications of the disclosure as illustrated herein, beingcontemplated as would normally occur to one skilled in the art to whichthe disclosure relates.

The present disclosure provides methods and compositions for thetreatment, identification, diagnosis, and prognosis of dystonia, ordystonia related disorders. In some embodiments, the disclosure providescompositions and methods for therapeutic intervention in subjectssuffering from dystonia, or dystonia related disorders. In otherembodiments, the disclosure provides methods and compositions fordiagnosis or prognosis of subjects who are suffering from, or are likelyto suffer from, dystonia, or dystonia related disorders. In stillfurther embodiments, the present disclosure provides methods fordiagnosing and treating subjects who are, or who may be, suffering fromdystonia.

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

Methods well known to those skilled in the art can be used to practiceembodiments of the present disclosure. See, for example, techniques asdescribed in Maniatis et al., 1989, MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, New York; Sambrook, J. et al., 2001, “MOLECULARCLONING: A LABORATORY MANUAL,” 3.sup.rd edition, Cold Spring HarborLaboratory Press. The contents of the above are incorporated in theirentirety herein by reference.

Additional methods well known to those skilled in the art can be used toprepare pharmaceutically acceptable compositions and methods oftreatment according to the present disclosure. See, for example, Goodman& Gilman, The Pharmacological Basis of Therapeutics, (11^(th) Edition)2005, McGraw-Hill. The contents of the above are incorporated in theirentirety herein by reference.

Additional methods well known to those skilled in the art can be usedfor therapeutic intervention in subjects with dystonia disorders. (Seee.g., Stacy, M., Ed., Physician's Desk Reference, Medical EconomicsCompany, Inc. Montvale, N. J. (54& Edition) 2000.)

Before describing the disclosed methods and compositions in detail, anumber of terms will be defined. As used herein, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise.

It is noted that terms like “preferably,” “commonly,” and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that can or cannot be utilized in a particular embodiment ofthis invention.

For the purposes of describing and defining this invention it is notedthat the term “substantially” is utilized herein to represent theinherent degree of uncertainty that can be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation can vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Definitions

In addition to terms defined elsewhere in the disclosure, the followingdefinitions apply to the description of the embodiments herein:

As used herein, the term “dystonia” refers to those neurologicalconditions and brain disorders characterized by often painful twistingmotions and postures and involuntary movements. Such conditions may beidiopathic, sporadic, or inherited forms with or without defined geneticcauses.

As used herein, the term “patient” or “subject” refers to mammals,including humans, animal pets, farm animals, zoo animals, and the like.Further, the patient or subject of the present disclosure may refer toany vertebrate species. In some embodiments, the patient or subject is ahuman. In certain embodiments, the subject is a human patient that is atrisk for, or suffering from, dystonia.

As used herein, the term “integrated stress response” refers to thecommon adaptive pathway eukaryotic cells activate in response to stressstimuli.

As used herein, “treatment,” “therapy” and/or “therapy regimen” refer tothe clinical intervention made in response to a disease, disorder orphysiological condition manifested by a patient or to which a patientmay be susceptible. The aim of treatment includes the alleviation orprevention of symptoms, slowing or stopping the progression or worseningof a disease, disorder, or condition and/or the remission of thedisease, disorder or condition.

The term “effective amount” or “therapeutically effective amount” refersto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results. As used herein an “effective” amount or a“therapeutically effective amount” of a pharmaceutical ingredient refersto a nontoxic but sufficient amount of the ingredient to provide thedesired effect. For example, one desired effect would be the preventionor treatment of dystonia or dystonia related disorders.

An amount that is “effective” according to embodiments of the disclosurewill vary from subject to subject, depending on the age and generalcondition of the individual, mode of administration, and the like. Thus,it is not always possible to specify an exact “effective amount.”However, an appropriate “effective” amount in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto. Pharmaceutically acceptable base addition saltscan be prepared from inorganic and organic bases. Salts derived frominorganic bases, include by way of example only, sodium, potassium,lithium, ammonium, calcium and magnesium salts. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary and tertiary amines.

As used herein, the term active pharmaceutical ingredient (API) means acompound or compounds with the ability to modulate dystonia or dystoniasymptoms in a subject in need thereof. In some embodiments, an API ofpresent disclosure is capable of modulating the levels or activity ofone or a plurality of genes or proteins selected from ATF4, BiP,eIF2-alpha, GADD34, and CreP.

As used herein, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativepolypeptide disclosed herein. An agonist can be any chemical compound,nucleic acid molecule, peptide or polypeptide can enhance activity of agene product (e.g., by stabilizing the gene product, preventing itsproteolytic degradation or increasing its enzymatic or binding activityor directly activating expression of a gene).

As used herein, the term “biological sample” or “biosample” or “sample”isolated from a subject includes, but is not limited to, a tissue orbodily fluid obtained from an animal, preferably a mammal and mostpreferably a human, containing tissues, cells, and/or biological fluidsisolated from a subject. Examples of biological samples include, but arenot limited to, tissues, cells, biopsies, blood, lymph, serum, plasma,cerebrospinal fluid, urine, feces, saliva, mucus and tears. In oneembodiment, the biological sample is a blood sample (such as a plasmasample) or biopsy sample (such as a tissue/cell sample). A biologicalsample may be obtained directly from a subject (e.g., by blood or tissuesampling) or from a third party (e.g., received from an intermediary,such as a healthcare provider or lab technician). In some embodiments, asample according to the disclosure is obtained from a human suspected ofhaving, or previously diagnosed as having, or in need of screening fordystonia. In certain embodiments, a biological sample is a sample ofblood or cerebrospinal fluid.

As used herein, the term “dystonia biomarker” refers to a naturallyoccurring biological molecule, or variant or mutation thereof, presentin a subject at varying concentrations useful in predicting the risk orincidence of a disease, the aggressiveness of a disease or a condition,or the likelihood of developing and/or surviving a disease or condition,such as dystonia. For example, the biomarker can be mis-localization ofa protein, such as AE Torsin a, that is present in in a subject ascompared to a control that indicates the presence of a disease, such asdystonia or likelihood of developing said disease/condition, such asdystonia.

As used herein “concentration” of a biomarker refers to both percentconcentration and absolute concentration of the biomarker. “Percentconcentration” refers to the comparative concentration of a biomarkerwith respect to another. “Absolute concentration” refers to a directmeasurement of the biomarker without comparison to other detectedspecies.

As used herein “pre-treatment level” or “pre-treatment range” refers toa level or concentration of one or more biomarkers (e.g. 1, 2, 3, 4, 5,or more biomarkers) in a biosample isolated from a subject beforeadministering treatment for a disorder characterized by dystonia, ordystonia related disorders. A pre-treatment level or pre-treatment rangeincludes, without limitation, an average of multiple measurements of thelevel or concentration of one or more biomarkers, or range of one ormore biomarkers, based on multiple measurements from a subject.

The term “quantify” or “quantification” may be used interchangeable, andrefer to a process of determining the quantity or abundance of asubstance in a sample (e.g., a biomarker), whether relative or absolute.For example, quantification may be determined by methods including butnot limited to, micro-array analysis, qRT-PCR, band intensity on aNorthern or Western blot, Fluorescent in situ hybridization (FISH), orby various other methods known in the art. Any method of detection failswithin the general scope of the present disclosure. The detectionmethods may be generic for the detection of proteins, phosphopeptides,nucleic acids, polypeptides and the like. The detection methods may bedirected towards the scoring of a presence or absence of one or morebiomarker molecules or may be useful in the detection of expressionlevels.

Methods of Treating or Preventing Dystonia

In embodiments, the present disclosure provides methods of treating asubject suffering from dystonia by administering one or moretherapeutically effective agents that modulate the integrated stressresponse. In some embodiments, the present disclosure provides methodsof treating a subject suffering from dystonia by administering one ormore therapeutically effective agents that modulate the intracellularsignaling pathway controlled by eIF2-alpha. In some embodiments, thepresent disclosure provides agents that modulate eIF2-alpha, eitherdirectly, or through modulation of other genes or proteins in theintracellular pathways of which eIF2-alpha is a component.

In some embodiments, methods of treating a subject suffering fromdystonia according to the present disclosure comprise treating a subjectwith one or more agents that increase the phosphorylation state of theeIF2-alpha protein. In further embodiments, the one or more agentscapable of increasing the phosphorylation state of eIF2-alpha comprisesan agent that increases the kinase activity of eIF2-alpha-specifickinases, thereby increasing the phosphorylation state of eIF2-alpha. Inother embodiments, the one or more agents capable of increasing thephosphorylation state of eIF2-alpha comprises an agent capable ofinhibiting eIF2-alpha-specific phosphatases, thereby increasing thephosphorylation state of eIF2-alpha. In still other embodiments, the oneor more agents capable of increasing the phosphorylation state ofeIF2-alpha comprises an agent that acts upstream of eIF2-alpha-specifickinases, or eIF2-alpha-specific phosphatases, thereby increasing orinhibiting, respectively, their kinase or phosphatase activity towardeIF2-alpha.

In some embodiments, the present disclosure provides agents that mimicphosphorylated eIF2-alpha. In other embodiments, the present disclosureprovides agents that mimic eIF2-alpha phosphorylated on Serine 51 of thehuman homolog of eIF2-alpha, or the corresponding residue on non-humanhomologs of eIF2-alpha.

In some embodiments, methods of treating a subject suffering fromdystonia according to the present disclosure comprise treating a subjectwith one or more agents that modulate the steady state levels ofeIF2-alpha protein in a cell. In certain embodiments, the one or moreagents that modulate the steady state levels of eIF2-alpha protein in acell regulate the expression of the eIF2-alpha gene, or the stability ofthe eIF2-alpha mRNA transcripts, or the rate of translation ofeIF2-alpha mRNA transcripts. In other embodiments, the one or moreagents that modulate the steady state levels of eIF2-alpha protein in acell modulate the stability or turnover of the eIF2-alpha protein in acell.

In some aspects, methods of treating a subject suffering from dystoniaaccording to the present disclosure comprise treating a subject with oneor more agents that modulate the phosphatase activity of the CrePprotein toward its targets. In embodiments, methods of the presentdisclosure comprise one or more agents that directly modulate CrePphosphatase activity. In other embodiments, methods of the presentdisclosure comprise one or more agents that indirectly modulate CrePphosphatase activity. In certain embodiments, the activity of CreP ismodulated at the level of protein or mRNA expression, such that thesteady state levels of CreP in the cell are increased or reduced. Incertain embodiments, the one or more agents that modulate the steadystate levels of CreP protein in a cell regulate the expression of theCreP gene, or the stability of the CreP mRNA transcripts, or the rate oftranslation of CreP mRNA transcripts. In other embodiments, the one ormore agents that modulate the steady state levels of CreP protein in acell modulate the stability or turnover of the CreP protein in a cell.In some embodiments, methods of the present disclosure comprise one ormore agents that modulate the phosphatase activity of CreP towardeIF2-alpha.

In further embodiments, the one or more agents capable of increasing thephosphorylation state of eIF2α according to the present method comprisessalubrinal, Sal-003, or guanabenz.

In some embodiments, the one or more agents capable of increasing thephosphorylation state of eIF2α according to the present method comprisesritonavir, liponavir, saquinavir, nelfinavir, and indinavir. Thoseskilled in the art will recognize that agents according to the presentdisclosure are capable of increasing the cellular stress response. See,e.g., Gassart et al., (2015) PNAS 113 (2) E117-E126, incorporated hereinin its entirety. Further, those skilled in the art will recognize thatagents of the present disclosure modulate the phosphorylation state ofthe eIF2-alpha protein.

In some aspects, methods of treating a subject suffering from dystoniaaccording to the present disclosure comprise treating a subject with oneor more agents that modulate the activity of the ATF4 protein. Inembodiments, methods of the present disclosure comprise one or agentsthat directly modulate ATF4 activity toward, for example, itstranscriptional targets. In other embodiments, methods of the presentdisclosure comprise one or more agents that indirectly modulate ATF4activity.

Accordingly, aspects of the disclosure provide compositions and methodsfor therapeutic intervention in subjects suffering from Dystonia orrelated disorders. In some embodiments, therapeutic interventioncomprises additional dystonia therapies known in the art. For example,therapies for dystonia may include, but are not limited to, thefollowing: (a) non-drug therapies, such as physical therapy,occupational therapy, speech and/or voice therapy, and relaxation/stressmanagement; (b) oral medications, such as anticholinergics (e.g.,trihexyophenidyl, benztmpine, ethopropazine, etc.), benzodiazepines(e.g., diazepam, clonazepam, lorazepam etc.), baclofen, dopaminergicagents/dopamine-depleting agents (e.g., levodopa, bromocriptine,clozapine, tetrabenazine, etc.), tetrabenezine and the like; (c)Botulinum Neurotoxin injections; (d) surgery, including deep brainstimulation, lesioning procedures (e.g., pallidotomy & Thalamotomy),peripheral surgeries, etc.) and complementary therapies, such asrelaxation techniques, yoga, pilates, biofeedback, acupuncture, and thelike. Such treatments are well known and particular to the patient andtype of dystonia and can be readily determined by one skilled in theart. In some embodiments, an active pharmaceutical ingredient, or API,of the present disclosure comprises an interfering molecule. As usedherein, the term “interfering molecule” refers to any molecule that iscapable of disrupting, or inhibiting, an intracellular signalingpathway. In preferred embodiments, the interfering molecule is capableof disrupting the signaling pathway. An interfering molecule of theinvention, for example, can inhibit the activity of a protein that isencoded by a gene either directly or indirectly. Direct inhibition canbe accomplished, for example, by binding to a protein and therebypreventing the protein from binding an intended target, such as areceptor. Indirect inhibition can be accomplished, for example, bybinding to a protein's intended target, such as a receptor or bindingpartner, thereby blocking or reducing activity of the protein.

Furthermore, an interfering molecule of the invention can inhibit a geneby reducing or inhibiting expression of the gene, inter alia byinterfering with gene expression (transcription, processing,translation, post-translational modification), for example, byinterfering with the gene's mRNA and blocking translation of the geneproduct or by post-translational modification of a gene product, or bycausing changes in intracellular localization.

Examples of suitable interfering molecules include, but are not limitedto, small molecules, antibodies, antisense RNAs, cDNAs,dominant-negative forms of molecules such as, without limitation, ATF4,BiP, eIF2-alpha, GADD34, and CreP, protein kinase inhibitors, proteaseinhibitors, combinations thereof; and the like.

In certain embodiments, an inhibitor of the disclosure can be a smallmolecule inhibitor. As used herein, the term “small molecule” refers toa molecule that has a molecular weight of less than about 1500 g/Mol. Asmall molecule can be, for example, small organic molecules, peptides orpeptide-like molecules. By way of example, a small molecule inhibitorsuitable in methods of the disclosure can be salubrinal, Sal-003, orguanabenz.

In certain embodiments, an inhibitor of the disclosure can be an HIVaspartyl protease inhibitor. Examples include, but are not limited to,ritonavir, liponavir, saquinavir, nelfinavir, and indinavir. In someembodiments, an inhibitor of the disclosure is selected from ritonavir,liponavir, saquinavir, and combination thereof.

In some embodiments, the inhibitor of the disclosure is a compound offormula

-   or pharmaceutically acceptable salts thereof, wherein-   each n is independently selected from 0, 1, 2, 3, and 4;-   R¹ is hydrogen or C₁-C₆ alkyl;-   R² and R³ are each independently selected from halogen, hydroxy,    C₁-C₆ alkyl, and C₁-C₆ alkoxy; and-   R⁴ is —C(O)R⁸, —C(O)OR⁸, —C(O)NHR⁸, or —C(O)N(C₁-C₆ alkyl)R⁸,    -   where R⁸ is C₁-C₆ alkyl, aryl, heteroaryl, heterocyclyl,        arylC₁-C₆ alkyl-, heteroarylC₁-C₆ alkyl-, or heterocyclylC₁-C₆        alkyl-, each optionally substituted with one or more of groups        selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy. C₁-C₆        alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino;-   R⁵ is selected from hydrogen and C₁-C₆ alkyl,-   or R⁵ and R⁸ together with atoms to which they are attached form    heterocyclyl optionally substituted with one or more of groups    selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆    alkoxy, amino, C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo, or thio;    and-   R⁶ is —Z—R⁷, wherein    -   Z is absent, —C₁-C₆ alkylene-, —O—C₁-C₆ alkylene-, —C₁-C₆        alkylene-O—, —NH—C₁-C₆ alkylene-, —C₁-C₆ alkylene-NH—, —O—,        —NH—, or —N(C₁-C₆ alkyl)-; and    -   R⁷ is aryl, heteroaryl, or heterocyclyl, each optionally        substituted with one or more of groups selected from halogen,        cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆        alkylamino, and diC₁-C₆ alkylamino.

In other embodiments, compound of formula (I) is of formula (I-A):

In other embodiments, the compound of formula (I) or formula (I-A) iswherein each n is 0. In some other embodiments, the compound of formula(I) or formula (I-A) is wherein R¹ is C₁-C₆ alkyl, or methyl, or ethyl,or, propyl, or ispopropyl.

In other embodiments, the compound of formula (I) or formula (I-A) iswherein R¹ is —C(O)R⁸, —C(O)OR⁸, —C(O)NHR⁸, or —C(O)N(C₁-C₆ alkyl)R⁸,where R¹ is C₁-C₆ alkyl, aryl, heteroaryl, heterocyclyl, arylC₁-C₆alkyl-, heteroarylC₁-C₆ alkyl-, or heterocyclylC₁-C₆ alkyl-, eachoptionally substituted with one or more of groups selected from halogen,cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆alkylamino, and diC₁-C₆ alkylamino; and/or R⁵ is selected from hydrogenand C₁-C₆ alkyl.

In some embodiments, the compound of formula (I) or formula (I-A) iswherein R⁴ is —C(O)NHR⁸ or —C(O)N(C₁-C₆ alkyl)R⁸, where R⁸ is arylC₁-C₆alkyl-, heteroarylC₁-C₆ alkyl-, or heterocyclylC₁-C₆ alkyl-, eachoptionally substituted with one or more of groups selected from halogen,cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆alkylamino, and diC₁-C₆ alkylamino. In some other embodiments, R⁵ ishydrogen.

In some embodiments, the compound of formula (I) or formula (I-A) iswherein —NR⁴R⁵ is of formula:

In other embodiments, the compound of formula (I) or formula (I-A) iswherein R⁵ and R⁸ together with atoms to which they are attached formheterocyclyl optionally substituted with one or more of groups selectedfrom halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino,C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo, or thio.

In some embodiments, the compound of formula (I) or formula (I-A) iswherein —NR⁴R⁵ is of formula:

In some embodiments, the compound of formula (I) or formula (I-A) iswherein Z is —C₁-C₆ alkylene-, —O—C₁-C₆ alkylene-, or —C₁-C₆alkylene-O—. In some other embodiments, R⁷ is aryl, heteroaryl, orheterocyclyl, each optionally substituted with one or more of groupsselected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy,amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino. In other embodiments, Zis —O-methylene- or -methylene-O—. In some other embodiments, R⁷ is arylor heteroaryl, each optionally substituted with one or more of groupsselected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy,amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino.

In some embodiments, the compound of formula (I) or formula (I-A) iswherein R⁶ is of formula:

In some embodiments, the inhibitor of the disclosure is a compound offormula

-   or pharmaceutically acceptable salts thereof, wherein-   m is selected from 0, 1, 2, 3, and 4;-   q is selected from 0, 1, 2, 3, and 4;-   X is —O—, —NH—, —CHR¹⁴—, —C(R¹⁴)₂—, or —CH₂—,-   R¹¹ is aryl, heteroaryl, or heterocyclyl, each optionally    substituted with one or more of groups selected from halogen, cyano,    nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino,    and diC₁-C₆ alkylamino;-   R¹² is —C(O)R⁸, —C(O)OR¹⁵, —C(O)NHR¹⁵, or —C(O)N(C₁-C₆ alkyl)R¹²,    -   where R⁵⁵ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl,        heteroaryl, heterocyclyl, arylC₁-C₆ alkyl-, heteroarylC₁-C₆        alkyl-, or heterocyclylC₁-C₆ alkyl-, each optionally substituted        with one or more of groups selected from halogen, cyano, nitro,        C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and        diC₁-C₆ alkylamino;-   R¹³ is each independently selected from halogen, hydroxy, C₁-C₆    alkyl, and C₁-C₆ alkoxy; and-   R¹⁴ is each independently selected from halogen, cyano, nitro, C₁-C₆    alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆    alkylamino;-   or two R¹⁴ with atoms to which they are attached form heterocyclyl    optionally substituted with one or more of groups selected from    halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino,    C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo, or thio.

In other embodiments, compound of formula (II) is of formula (II-A):

In other embodiments, the compound of formula (II) or formula (II-A) iswherein each m is 0.

In some embodiments, the compound of formula (II) or formula (II-A) iswherein R¹¹ is aryl or heteroaryl, each optionally substituted with oneor more of groups selected from halogen, cyano, nitro, C₁-C₆ alkyl,hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino.In some other embodiments, the compound of formula (II) or formula(II-A) is wherein R¹¹ is aryl or heteroaryl. In some other embodiments,the compound of formula (II) or formula (II-A) is wherein R¹¹ isheteroaryl.

In some embodiments, the compound of formula (II) or formula (II-A) iswherein R¹¹ is quinolinyl, or quinolin-2-yl.

In some embodiments, the compound of formula (II) or formula (II-A) iswherein R¹² is —C(O)NHR¹⁵ or —C(O)N(C₁-C₆ alkyl)R¹⁵, where R¹⁵ is C₁-C₆alkyl, C₂-C₆ alkenyl, C₁-C₆ alkynyl, aryl, heteroaryl, heterocyclyl,arylC₁-C₆ alkyl-, heteroarylC₁-C₆ alkyl-, or heterocyclylC₁-C₆ alkyl-,each optionally substituted with one or more of groups selected fromhalogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆alkylamino, and diC₁-C₆ alkylamino.

In some embodiments, the compound of formula (TI) or formula (TI-A) iswherein R¹² is —C(O)NHR¹⁵ or —C(O)N(C₁-C₆ alkyl)R¹⁵, where R¹⁵ is C₁-C₆alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In some embodiments, the compound of formula (II) or formula (II-A) iswherein R¹² is —C(O)NHR¹⁵ or —C(O)N(C₁-C₆ alkyl)R⁸, where R¹⁵ is C₁-C₆alkyl. In some other embodiments, R¹² is —C(O)NHR¹⁵ and R¹⁵ is C₁-C₆alkyl. In some other embodiments, R¹² is —C(O)NH(tert-butyl).

In some embodiments, the compounds of formula (II) or formula (II-A) iswherein q is 1, —CHR⁴—, and two R¹⁴ with atoms to which they areattached form heterocyclyl optionally substituted with one or more ofgroups selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆alkoxy, amino, C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo, or thio. Inother embodiments, two R¹⁴ with atoms to which they are attached formdecahydro-isoquinolin-2-yl. In other embodiments, two R¹⁴ with atoms towhich they are attached form 3-R¹²-decahydro-isoquinolin-2-yl.

In some embodiments, an inhibitor according to the disclosure can be,for example, a small molecule inhibitor, an antibody, a nucleic acidsuch as an antisense nucleic acid, a short interfering RNA (siRNA)molecule, or a short hairpin RNA (shRNA) molecule. In addition, suchinhibitors can be specifically designed using the methods describedherein or using methods known in the art. For example, antibodies,particularly neutralizing antibodies and preferably monoclonalantibodies, can be generated by conventional means as described, forexample, in “Antibodies: A Laboratory Manual” by Harlow and Lane (ColdSpring Harbor Press, 1988), which is hereby incorporated by reference.

In further embodiments, inhibitors or the disclosure are species ofshort interfering RNA (siRNA). The term “short interfering RNA” or“siRNA” as used herein refers to a double stranded nucleic acid moleculecapable of RNA interference or “RNAi”, as disclosed, for example, inBass, 2001, Nature 411: 428-429; Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al., International PCT Publication No. WO00/44895; Zernicka-Goetz et al., International PCT Publication No. WO01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetincket al., International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914. As used herein, siRNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically modified nucleotides andnon-nucleotides having RNAi capacity or activity.

In still other embodiments, an API according to the present disclosurecomprises an agonist. An agonist of the invention can increase theactivity of a protein that is encoded by a gene either directly orindirectly. Direct activation can be accomplished, for example, bybinding to a protein and thereby enhancing binding of the protein to anintended target, such as a receptor. Indirect activation can beaccomplished, for example, by binding to a protein's intended target,such as a receptor or binding partner, and enhancing activity, e.g. byincreasing the effective concentration of the target. Furthermore, anagonist of the invention can activate a gene by increasing expression ofthe gene, e.g., by increasing gene expression (transcription,processing, translation, post-translational modification), for example,by stabilizing the gene's mRNA or blocking degradation of the mRNAtranscript, or by post-translational modification of a gene product, orby causing changes in intracellular localization.

Suitable agonist molecules specifically include agonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, small organic molecules, etc. Methods foridentifying agonists of a native polypeptide may comprise contacting anative polypeptide with a candidate agonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the native polypeptide.

Methods of Detecting Dystonia in a Subject

One aspect of the present disclosure provides a method of determiningwhether a subject is suffering from dystonia comprising determining thepresence of a dystonia biomarker in a biological sample derived from thesubject, wherein the presence of the biomarker is associated withdystonia.

In embodiments, the present disclosure provides methods of determiningthe presence of dystonia in a subject, and/or the likelihood of asubject developing dystonia, by measuring the level or activity of oneor a plurality of biomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) ina subject. In embodiments, the method comprises obtaining a sample froma subject to measure the one or a plurality of biomarkers in the sample.The subject may be any mammal, such as a human.

In embodiments, the methods of the disclosure may comprise obtainingmore than one sample, such as two samples, three samples, four samples,or more, from one or more subjects. In certain embodiments, the methodsof the disclosure comprise comparing the expression of one or aplurality of biomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) fromone or more subjects. In alternative embodiments, methods of thedisclosure compare a one or a plurality of biomarkers in a single sampleagainst a “standardized,” or “reference,” level of one or a plurality ofbiomarkers capable of detecting dystonia or dystonia related disorders.Before analyzing a sample according to the disclosure, in someembodiments one or more sample preparation operations, or steps, areperformed upon the sample. Typically, these sample preparationoperations, or steps, comprise one or more treatments, such as, withoutlimitation, concentration, suspension, extraction of intracellularmaterial, e.g., proteins/phosphopeptides from tissue/whole cell samples,and the like. Any method required for the processing of a sample priorto detection by any of the methods noted herein falls within the scopeof the present disclosure. These methods are typically well known by aperson skilled in the art.

In some aspects, the disclosure provides one or more biomarkers (e.g. 1,2, 3, 4, 5, or more biomarkers) associated with dystonia comprisingcomponents of an intracellular signaling pathway mediated by eIF2-alpha.In embodiments, the one or more biomarkers associated with dystoniacomprise the expression level of one or more components of anintracellular signaling pathway mediated by eIF2-alpha. In otherembodiments, the one or more biomarkers associated with dystoniacomprise an activity level of one or more components of an intracellularsignaling pathway mediated by eIF2-alpha. In still other embodiments,the one or more biomarkers associated with dystonia comprise mutationsin one or more components of an intracellular signaling pathway mediatedby eIF2-alpha. In some embodiments, the biomarker(s) associated withdystonia comprise, one or more of the following proteins: Torsin1a,ATF4, BiP, eIF2-alpha, GADD34, CreP, HERPUD1, RAP1A, and MAP2K5,variants and mutations thereof, and combinations thereof. In certainembodiments, presence of a AE Torsin1a mis-localization associated withan increased likelihood of developing dystonia. In other embodiments,the presence of a AE Torsin1a mis-localization is associated with thepresence of dystonia in a subject.

In some embodiments, the one or more biomarkers (e.g. 1, 2, 3, 4, 5, ormore biomarkers) associated with Dystonia comprise mutations incomponents of intracellular signaling pathways mediated by theeIF2-alpha protein. In other embodiments, the one or more biomarkersassociated with Dystonia comprise mutations in components ofintracellular signaling pathways mediated by the ATF4 protein. In stillfurther embodiments, the one or more biomarkers associated with Dystoniacomprise mutations in components of intracellular signaling pathwaysmediated by the CReP protein. In yet additional embodiments, the one ormore biomarkers associated with Dystonia comprise mutations incomponents of intracellular signaling pathways that are upstream ordownstream of any of eIF2-alpha, ATF4, or CreP.

In some embodiments, the disclosure provides one or more biomarkers thatindicate the state of an intracellular signaling pathway, such as anactivated or inhibited intracellular signaling pathway. In certainembodiments, the disclosure provides one or more biomarkers thatindicate the signaling state of the intracellular signaling pathwaymediated by eIF2-alpha. In some embodiments, the disclosure provides oneor more biomarkers comprising the phosphorylation state of, e.g., theExtracellular Signal-Regulated Kinases (ERKs), eIF2-alpha, eIF2-alphakinases, or eIF2-alpha phosphatases. In other embodiments, thedisclosure provides one or more biomarkers comprising thephosphorylation state of intracellular proteins downstream of eIF2-alphasignaling, such as the phosphorylation state of ATF4. In still furtherembodiments, the disclosure provides one or more biomarkers comprisingthe phosphorylation state of tyrosine 37 in ATF4.

In certain embodiments, the one or more biomarkers (e.g. 1, 2, 3, 4, 5,or more biomarkers) associated with Dystonia comprise mutations in theATF4 protein found in sporadic dystonia patients. In a particularembodiment, the biomarker associated with Dystonia comprises a prolineto leucine substitution at position 46 of the wild-type ATF4 proteinsequence. In another particular embodiment, the biomarker associatedwith Dystonia comprises a tyrosine to phenylalanine substitution atposition 37 of the wild-type ATF4 protein sequence. In still anotherparticular embodiment, the biomarker associated with Dystonia comprisesan arginine to lysine substitution at position 296 of the wild-type ATF4protein sequence. In yet another particular embodiment, the biomarkerassociated with Dystonia comprises an aspartic acid to tyrosinesubstitution at position 35 of the wild-type ATF4 protein sequence. Instill further embodiments, the biomarker associated with Dystoniacomprises an aspartic acid to asparagine substitution at position 35 ofthe wild-type ATF4 protein sequence. In still further embodiments, thebiomarker associated with Dystonia comprises an methionine to valinesubstitution at position 1 of the wild-type ATF4 protein sequence

In still further embodiments, the biomarkers (e.g. 1, 2, 3, 4, 5, ormore biomarkers) associated with Dystonia according the presentdisclosure comprise the expression of one or more genes responsive tothe ATF protein. In some embodiments, the biomarkers of the presentdisclosure comprise reduced expression of one or more genes responsiveto the ATF protein. In other embodiments, the biomarkers of the presentdisclosure comprise increased expression of one or more genes responsiveto the ATF protein.

Another aspect of the present disclosure provides a method of predictingthe likelihood of a subject developing dystonia comprising, consistingof, or consisting essentially of quantifying the amount of a dystoniabiomarker present in a biological sample derived from the subject,wherein the present of the biomarker is associated with a likelihood ofthe subject developing dystonia.

Another aspect of the present disclosure provides a method ofdetermining whether a subject is suffering from dystonia comprising,consisting of, or consisting essentially of: (a) obtaining a biologicalsample from a subject; (b) determining the expression level of adystonia biomarker in the biological sample; (c) comparing theexpression level of the biomarker in the biological sample with that ofa control, wherein the presence of the biomarker in the sample that isin an amount greater than that of the control indicates the subject issuffering from dystonia; and (d) administering appropriate anti-dystoniatherapy if the biomarker is expressed.

Another aspect of the present disclosure provides a method ofdetermining whether a subject is at risk of developing dystoniacomprising, consisting of, or consisting essentially of (a) obtaining abiological sample from a subject; (b) determining the expression levelof a dystonia biomarker in the biological sample; (c) comparing theexpression level of the biomarker in the biological sample with that ofa control, wherein the presence of the bio marker in the sample that isin an amount greater than that of the control indicates the subject isat risk of developing dystonia; and (d) administering appropriateanti-dystonia therapy if the biomarker is expressed.

In other aspects, the disclosure provides methods for monitoringprogression of dystonia in a patient, for example, to determine if apatient is responding positively or negatively to a certain treatmentregime.

In another embodiment, the determination of the presence of dystonia ina subject and/or the likelihood of a person developing dystonia can bedetermined by comparing the subject's biomarker profile to a referencebiomarker profile, such as one that corresponds to biological samplesobtained from a normal population that do not have a condition such asdystonia, or that corresponds to biological samples obtained from apopulation that have a condition such as dystonia. As used herein, a“reference biomarker profile” means a control level or range obtainedfrom multiple subjects, or from one or more subjects over time.Optionally, a reference profile according to the disclosure comprisesmultiple biomarker expression profiles, with each corresponding to adifferent stage of a condition such as dystonia. Optionally, a referenceprofile according to the disclosure comprises a reference biomarkerprofile from one or more subjects.

In some embodiments, the level or concentration of one or morebiomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) in a subject isincreased relative to a reference biomarker profile. In otherembodiments, the level or concentration of one or more biomarkers isdecreased relative to a reference biomarker profile. In still otherembodiments, the level or concentration of one or more biomarkers isdecreased, whereas the level or concentration of other biomarkers areincreased, relative to a control level or range.

In embodiments, the level or concentration of one or more biomarkers(e.g. 1, 2, 3, 4, 5, or more biomarkers) changes by at least about 10percent, for example, by at least about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 100 percent, relative to a control levelor range. In some embodiments, the level or concentration of one or morebiomarkers changes by at least about 2-fold, for example, at least about4, 6, 8, 10, 20, 40, 60, 80, or 100 fold, relative to a referencebiomarker profile. A “control level” or “reference level” as used hereinrefers to an amount or range of amounts of a biochemical marker, suchas, without limitation, Torsin1a, ATF4, BiP, eIF2-alpha, GADD34, CreP,HERPUD1, RAP1A, and MAP2K5, variants and mutations thereof, found in acomparable biosample in subjects not suffering from dystonia, or from asubject known to be suffering from dystonia, or from a subject with aknown inherited or genetic form of dystonia. The “control level” or“reference level” can also be based on a database of biochemical markerssuch as from previously tested subjects who did not exhibit, or develop,dystonia or dystonia related disorders over a clinically relevant time.

In some embodiments, the present disclosure provides one or a pluralityof biomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) capable ofdetermining the presence of dystonia in a subject. In furtherembodiments, the disclosure provides one or a plurality of biomarkerscapable of determining the likelihood of a subject developing dystonia.

In certain embodiments, the disclosure provides biomarkers (e.g. 1, 2,3, 4, 5, or more biomarkers), such as Torsin1a, ATF4, BiP, eIF2-alpha,GADD34, CreP, HERPUD1, RAP1A, and MAP2K5, variants and mutations thereofas biomarkers useful for determining the presence of dystonia in asubject, and/or the likelihood of a subject developing dystonia. Theinventors have determined that certain biomarkers are directly involvedin dystonia and/or likelihood of developing dystonia, and theirexpression pattern in a biological sample can be associated with thepathophysiological status of the subject suffering from dystonia.

The disclosure also provides methods for identifying a subject that iseligible for reimbursement of an insurance claim for treatment ofdystonia, or dystonia related disorders. In these embodiments, themethods comprise the steps of: (a) isolating a biosample from a subject;(b) determining a level or concentration of one or more biomarkerspresent in the biosample; and (c) as eligible for reimbursement of theinsurance claim when the concentration of one or more biomarkers isincreased or decreased relative to an insurance control value. In theseembodiments, the insurance control value refers to an amount or range ofamounts of a biochemical marker.

The insurance control value refers to an amount or range of amounts ofone or more biochemical markers found in a comparable biosample insubjects not suffering from dystonia, or dystonia related disorders, andused as an insurance reimbursement criterion by, inter alia, a healthinsurer. In another embodiment, insurance coverage of an individual isassessed as a function of actuarial data that is obtained fromindividuals with changes in concentration of the one or more biomarkersdisclosed herein. A control level according to embodiments of thepresent methods is based on a database of biochemical marker suchcomprising one or more biomarkers from previously tested subjects whodid not exhibit or develop dystonia, or dystonia related disorders, overa clinically relevant time frame. Additionally, a control levelaccording to embodiments of the present methods is based on anindividual that did not file a reimbursement claim based on dystonia, ordystonia related disorders, within an actuarially relevant time period.

In some aspects, a subject is included or enrolled in an insurance planbased on the insurable status of the subject or wherein the rate or costof the insurance is based on the insurable status of the subject.Alternatively, the subject is excluded from an insurance plan based onthe insurable status of the subject. In some such instances, anorganization that provides medical insurance requests or otherwiseobtains information concerning a subject's biochemical marker status anduses that information to determine an appropriate medical insurancepremium or reimbursement of an insurance claim relating to treatment ofthe subject.

The disclosure also provides methods for determining the efficacy of atreatment for dystonia, or dystonia related disorders, in a subject. Inthese embodiments, the methods comprise the steps of: (a) treating asubject for dystonia, or a dystonia related disorder; (b) isolating abiosample from the subject; (c) determining a level or concentration ofone or more biomarkers present in the biosample; and (d) determining theefficacy of the treatment for dystonia, or dystonia related disorders,when the concentration of one or more biomarkers is increased ordecreased relative to a pre-treatment level or pre-treatment range ofthe one or more biomarkers. In embodiments, the one or more biomarkerscomprise the genes, or gene products, selected from Torsin1a, ATF4, BiP,eIF2-alpha, GADD34, CreP, HERPUD1, RAP1A, and MAP2K5. In someembodiments, the control sample is a biological sample from a normalsubject, i.e. an individual without dystonia, or dystonia relatedsymptoms, or one who responds to therapy for a condition characterizedby dystonia, or dystonia related disorders.

In some embodiments, a panel of biomarkers capable of predicting theoccurrence of dystonia, or dystonia related disorders, or determiningthe efficacy of a treatment for dystonia, or dystonia related disordersis provided. Embodiments of a biomarker panel are comprised of two ormore biomarkers. In one embodiment, a biomarker panel comprises thegenes, or gene products of, two or more of Torsin1a, ATF4, BiP,eIF2-alpha, GADD34, CreP, HERPUD1, RAP1A, and MAP2K5. In someembodiments, a panel of two or more biomarkers disclosed herein providean improved method according to the disclosure. In embodiments,improvements using a panel of two or more biomarkers comprise greateraccuracy or specificity relative to a single biomarker, or to analternative biomarker panel.

As described above, certain embodiments of the disclosure comprisedetecting one or more biomarkers (e.g. 1, 2, 3, 4, 5, or morebiomarkers) in a sample. In some embodiments, detection may comprisedetecting the presence versus absence of one or more biomarkers. In someembodiments, detection may comprise quantifying the level or degree ofexpression of one or more biomarkers, depending on the method ofdetection employed. Determining the amount of a biomarker, such asTorsin1a, ATF4, BiP, eIF2-alpha, GADD34, CreP, HERPUD1, RAP1A, andMAP2K5 in a sample relates to measuring an amount or concentration ofbiomarker protein or nucleic acid in the sample. In certain embodiments,such measurements are semi-quantitative or quantitative. Measuring canbe done directly or indirectly. Direct measuring relates to measuringthe amount or concentration of the protein or nucleic acid based on asignal which is obtained from the protein or nucleic acid itself and theintensity of which directly correlates with the number of molecules ofthe protein or nucleic acid present in the sample. Such asignal—sometimes referred to as intensity signal—may be obtained, forexample, by measuring an intensity value of a specific physical orchemical property of the protein or nucleic acid. Indirect measuringincludes measuring of a signal obtained from a secondary component(i.e., a component not being the protein or nucleic acid itself) or abiological read out system, e.g., measurable cellular responses,ligands, labels, or enzymatic reaction products.

In aspects of the present disclosure, determining the amount of apeptide or polypeptide (protein) can be achieved by all known means fordetermining the amount of a peptide or polypeptide in a sample. Forexample, without limitation, the methods of the present disclosurecomprise immunoassay devices and methods that may utilize labelledmolecules in various sandwich, competition, or other assay formats. Saidassays will develop a signal which is indicative for the presence orabsence of the peptide or polypeptide. Moreover, the signal strength canbe correlated directly or indirectly (e.g., reverse-proportional) to theamount of polypeptide present in a sample. Other suitable methodscomprise measuring a physical or chemical property specific for thepeptide or polypeptide such as its precise molecular mass or NMRspectrum. Said methods may comprise biosensors, optical devices coupledto immunoassays, biochips, and analytical devices such asmass-spectrometers, NMR-analyzers, or chromatography devices. Othersuitable methods include micro-plate ELISA-based methods,fully-automated or robotic immunoassays.

In other embodiments, the amount of a peptide or polypeptide isdetermined by contacting the peptide with a specific ligand, optionallyremoving non-bound ligand, and measuring the amount of bound ligand. Thebound ligand will generate an intensity signal. Binding according to thepresent disclosure includes both covalent and non-covalent binding. Aligand according to the present disclosure can be any compound, e.g., apeptide, polypeptide, nucleic acid, or small molecule, binding to thepeptide or polypeptide described herein. Suitable ligands includeantibodies, nucleic acids, peptides or polypeptides such as receptors orbinding partners for the peptide or polypeptide and fragments thereofcomprising the binding domains for the peptides, and aptamers, e.g.,nucleic acid or peptide aptamers. Methods to prepare such ligands arewell known in the art. For example, identification and production ofsuitable antibodies or aptamers is offered by commercial suppliers.Those skilled in the art are familiar with methods to developderivatives of such ligands with higher affinity or specificity. Forexample, random mutations can be introduced into the nucleic acids,peptides, or polypeptides. These derivatives can then be tested forbinding according to screening procedures known in the art, e.g., phagedisplay. Antibodies as referred to herein include both polyclonal andmonoclonal antibodies, as well as fragments thereof, such as Fv, Fab andF(ab)₂ fragments that are capable of binding antigen or hapten. Thepresent disclosure also includes single chain antibodies and humanizedhybrid antibodies wherein amino acid sequences of a non-human donorantibody exhibiting a desired antigen-specificity are combined withsequences of a human acceptor antibody. The donor sequences will usuallyinclude at least the antigen-binding amino acid residues of the donorbut may comprise other structurally and/or functionally relevant aminoacid residues of the donor antibody as well. Such hybrids can beprepared by several methods well known in the art. In some embodiments,the ligand or agent specifically binds to the peptide or polypeptide.Specific binding according to the present disclosure means that theligand or agent should not bind substantially to (“cross-react” with)another peptide, polypeptide, or substance present in the sample to beanalyzed. In certain embodiments, the specifically bound peptide orpolypeptide should be bound with at least 3 times higher, at least 10times higher, or at least 50 times higher affinity than any otherrelevant peptide or polypeptide. Non-specific binding may be tolerableif it can still be distinguished and measured unequivocally, e.g.,according to its size on a Western Blot, or by its relatively higherabundance in the sample. Binding of the ligand can be measured by anymethod known in the art. In certain embodiments, the method issemi-quantitative or quantitative.

The amount of a peptide or polypeptide may also be determined bycontacting a solid support comprising a ligand for the peptide orpolypeptide as specified above with a sample comprising the peptide orpolypeptide and measuring the amount peptide or polypeptide which isbound to the support. The ligand may be chosen from the group consistingof nucleic acids, peptides, polypeptides, antibodies and aptamers, andcan be present on a solid support in immobilized form. Materials formanufacturing solid supports are well known in the art and include,inter alia, commercially available column materials, polystyrene beads,latex beads, magnetic beads, colloid metal particles, glass and/orsilicon chips and surfaces, nitrocellulose strips, membranes, sheets,duracytes, wells and walls of reaction trays, and plastic tubes. Theligand or agent may be bound to many different carriers. Examples ofwell-known carriers include glass, polystyrene, polyvinyl chloride,polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insolublefor the purposes of the disclosure. Suitable methods forfixing/immobilizing said ligand are well known and include, but are notlimited to ionic, hydrophobic, covalent interactions and the like. It isalso contemplated to use “suspension arrays” as arrays according to thepresent disclosure (Nolan et al., 2002, Trends Biotechnol. 20(1): 9-12).In such suspension arrays, the carrier, e.g., a microbead ormicrosphere, is present in suspension. The array consists of differentmicrobeads or microspheres, possibly labelled, carrying differentligands. Methods of producing such arrays, for example based onsolid-phase chemistry and photo-labile protective groups, are generallyknown (U.S. Pat. No. 5,744,305).

In some embodiments, a peptide or polypeptide of the disclosure ismeasured directly, e.g., by NMR or surface plasmon resonance. In otherembodiments, an enzymatic reaction product may be measured (e.g., theamount of a protease can be measured by measuring the amount of cleavedsubstrate, e.g., on a Western Blot). Alternatively, the peptide orpolypeptide may exhibit enzymatic properties itself, and may becontacted with a suitable substrate allowing detection by the generationof an intensity signal. For measurement of enzymatic reaction products,the amount of substrate can be saturating. The substrate may also belabelled with a detectable label prior to the reaction. In oneembodiment, a sample is contacted with the substrate for an adequateperiod of time. An adequate period of time refers to the time necessaryfor a detectable and measurable amount of product to be produced.Instead of measuring the amount of product, the time necessary forappearance of a given (e.g., detectable) amount of product can bemeasured.

In still other embodiments, a peptide or polypeptide of the disclosureis measured indirectly, and may be coupled covalently or non-covalentlyto a label allowing detection and measurement. Labelling may be done bydirect or indirect methods. Direct labelling involves coupling of thelabel directly (covalently or non-covalently) to the peptide orpolypeptide. Indirect labelling involves binding (covalently ornon-covalently) of a secondary ligand to the peptide or polypeptide. Thesecondary ligand should specifically bind to the peptide or polypeptide.Said secondary ligand may be coupled with a suitable label and/or be thetarget (receptor) of tertiary ligand binding to the secondary ligand.Secondary, tertiary, or even higher order ligands are often used toincrease the signal. Suitable secondary and higher order ligands mayinclude antibodies, secondary antibodies, and the well-knownstreptavidin-biotin system (Vector Laboratories, Inc.). The peptide orpolypeptide or substrate may also be “tagged” with one or more tags asknown in the art. Such tags may then be targets for higher orderligands. Suitable tags include biotin, digoxygenin, His-Tag,Glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virushemagglutinin (HA), maltose binding protein, and the like. In the caseof a peptide or polypeptide, the tag may be located at the N-terminusand/or C-terminus. Suitable labels are any labels detectable by anappropriate detection method. Typical labels include gold particles,latex beads, acridan ester, luminol, ruthenium, enzymatically activelabels, radioactive labels, magnetic labels (e.g., magnetic beads,including paramagnetic and superparamagnetic labels), and fluorescentlabels. Enzymatically active labels include e.g., horseradishperoxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, andderivatives thereof. Suitable substrates for detection includedi-amino-benzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP(4-nitro blue tetrazolium chloride and5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stocksolution from Roche Diagnostics), CDP-STAR™ (Amersham Biosciences),ECFTe (Amersham Biosciences). A suitable enzyme-substrate combinationmay result in a colored reaction product, fluorescence, orchemoluminescence, which can be measured according to methods known inthe art (e.g., using a light-sensitive film or a suitable camerasystem). As for measuring the enzymatic reaction, the criteria givenabove apply analogously. Typical fluorescent labels include fluorescentproteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red,Fluorescein, and the Alexa dyes (e.g., Alexa 568). Other fluorescentlabels are available e.g., from Molecular Probes (Oregon). Also the useof quantum dots as fluorescent labels is contemplated. Typicalradioactive labels include ³⁵S, ¹²⁵I, ³²P, ³³P, and the like. Aradioactive label can be detected by any method known and appropriate,e.g., a light-sensitive film or a phosphor imager. Suitable measurementmethods according the present disclosure also include precipitation(particularly immunoprecipitation), electrochemiluminescence(electro-generated chemiluminescence), RIA (radioimmunoassay), ELISA(enzyme-linked immunosorbent assay), sandwich enzyme immune tests,electrochemiluminescence sandwich immunoassays (ECLIA),dissociation-enhanced lanthanide fluoro 31 hosph assay (DELFIA),scintillation proximity assay (SPA), turbidimetry, nephelometry,latex-enhanced turbidimetry or nephelometry, or solid phase immunetests. Other methods known in the art (such as gel electrophoresis, 2Dgel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE),Western Blotting, and mass spectrometry) can be used alone or incombination with labelling or other detection methods as describedabove.

In some embodiments, the detection methods of the disclosure comprise insitu methods. In alternative embodiments, the detection methods of thedisclosure comprise screening methods.

As used herein, an in situ method refers to the detection of protein,phosphopeptide, and/or nucleic acid molecules in a sample wherein thestructure of the sample has been preserved. This may thus be a biopsy(e.g., a tissue biopsy) wherein the structure of the tissue ispreserved. In situ methods are generally histological i.e. microscopicin nature and include but are not limited to methods such as: in situhybridization techniques and in situ PCR methods.

In some embodiments, methods of the disclosure comprise in situhybridization methods. In situ hybridization (ISH) applies andextrapolates the technology of nucleic acid and/or polypeptidehybridization to the single cell level, and, in combination with the artof cytochemistry, immunocytochemistry and immunohistochemistry, permitsthe maintenance of morphology and the identification of cellular markersto be maintained and identified, allows the localization of sequences tospecific cells within populations, such as tissues and blood samples.ISH is a type of hybridization that uses a complementary nucleic acid tolocalize one or more specific nucleic acid sequences in a portion orsection of tissue (In situ), or, if the tissue is small enough, in theentire tissue (whole mount ISH). DNA ISH can be used to determine thestructure of chromosomes and the localization of individual genes andoptionally their copy numbers. Fluorescent DNA ISH (FISH) can forexample be used in medical diagnostics to assess chromosomal integrity.RNA ISH is used to assay expression and gene expression patterns in atissue/across cells, such as the expression of miRNAs/nucleic acidmolecules. Sample cells are treated to increase their permeability toallow the probe to enter the cells, the probe is added to the treatedcells, allowed to hybridize at pertinent temperature, and then excessprobe is washed away. A complementary probe is labeled with aradioactive, fluorescent or antigenic tag, so that the probe's locationand quantity in the tissue can be determined using autoradiography,fluorescence microscopy or immunoassay, respectively. The sample may beany sample as herein described. The probe is likewise a probe accordingto any probe based upon the biomarkers mentioned herein.

In some embodiments, methods of the disclosure comprise in situ PCR. Insitu PCR comprises PCR based amplification of target nucleic acidsequences prior to ISH. For detection of RNA, an intracellular reversetranscription (RT) step is introduced to generate complementary DNA fromRNA templates prior to in situ PCR. This enables detection of low copyRNA sequences. Prior to in situ PCR, cells or tissue samples are fixedand permeabilized to preserve morphology and permit access of the PCRreagents to the intracellular sequences to be amplified. PCRamplification of target sequences is performed either in intact cellsheld in suspension or directly in cytocentrifuge preparations or tissuesections on glass slides. In the former approach, fixed cells suspendedin the PCR reaction mixture are thermally cycled using conventionalthermal cyclers. After PCR the cells are cytocentrifugated onto glassslides with visualization of intracellular PCR products by ISH orimmunohistochemistry. In situ PCR on glass slides is performed byoverlaying the samples with the PCR mixture under a coverslip which isthen sealed to prevent evaporation of the reaction mixture. Thermalcycling is achieved by placing the glass slides either directly on topof the heating block of a conventional or specially designed thermalcycler or by using thermal cycling ovens. Detection of intracellularPCR-products is achieved by one of two entirely different techniques. Inindirect in situ PCR by ISH with PCR-product specific probes, or indirect in situ PCR without ISH through direct detection of labelednucleotides (e.g. digoxigenin-11-dUTP, fluorescein-dUTP, ³H-CTP orbiotin-16-dUTP) which have been incorporated into the PCR productsduring thermal cycling.

In some embodiments, the disclosure provides one or more biomarkerscomprising a radiolabel facilitating medical diagnostic procedures,including Positron Emission Tomography (PET) and Single Photon EmissionComputed Tomography (SPECT). PET and SPECT are very sensitive techniquesand require small quantities of radiolabeled compounds, called tracers.The labeled compounds, such as a biomarker according to the disclosure,are transported, accumulated, and converted in vivo in exactly the sameway as the corresponding non-radioactively labeled compounds. Tracers,or probes, can be radiolabeled with a radionuclide useful for PETimaging, such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶¹Cu, ⁶²C, ⁶⁴C, ⁶⁷Cu, ⁶⁸Ga, ¹²⁴Iand ¹³¹I, or with a radionuclide useful for SPECT imaging, such as ⁹⁹Tc,⁷⁵Br, ⁶¹Cu, ¹³Gd, ¹²⁵I, ¹³¹I and ³²P. One example of a PET probe is[¹⁸F]-fluorodeoxyglucose ([¹⁸F]-FDG).

Screening methods, as used herein, employ techniques of molecularbiology. In embodiments, the screening methods of the disclosurecomprise preparing sample material in order to access the nucleic acidand/or polypeptide molecules to be detected. Screening methods include,but are not limited to methods such as: Array systems, affinitymatrices, Northern blotting and PCR techniques, such as real-timequantitative RT-PCR.

In some embodiments, the methods of the disclosure comprise screeningone or more samples using, for example, a microarray. A microarray is amicroscopic, ordered array of nucleic acids, proteins, small molecules,cells or other substances that enables parallel analysis of complexbiochemical samples. A DNA microarray consists of different nucleic acidprobes, known as capture probes that are chemically attached to a solidsubstrate, which can be a microchip, a glass slide or amicrosphere-sized bead. Microarrays can be used e.g. to measure theexpression levels of large numbers of polypeptides/proteins/nucleicacids simultaneously.

Several types of microarrays can be employed in embodiments of thedisclosure, such as, without limitation, spotted oligonucleotidemicroarrays, pre-fabricated oligonucleotide microarrays or spotted longoligonucleotide arrays.

In spotted oligonucleotide microarrays the capture probes areoligonucleotides complementary to nucleic acid sequences. This type ofarray is typically hybridized with amplified. PCR products ofsize-selected small RNAs from two samples to be compared that arelabelled with two different fluorophores. Alternatively, total RNAcontaining the small RNA fraction is extracted from the abovementionedtwo samples and used directly without size-selection of small RNAs, and3′ end labeled using T4 RNA ligase and short RNA linkers labelled withtwo different fluorophores. The samples can be mixed and hybridized toone single microarray that is then scanned, allowing the visualizationof up-regulated and down-regulated biomarker genes in one go. Thedownside of this is that the absolute levels of gene expression cannotbe observed, but the cost of the experiment is reduced by half.Alternatively, a universal reference can be used, comprising of a largeset of fluorophore-labelled oligonucleotides, complementary to the arraycapture probes.

Spotted long oligonucleotide arrays are composed of 50 to 70-meroligonucleotide capture probes, and are produced by either ink-jet orrobotic printing. Short Oligonucleotide Arrays are composed of 20-25-meroligonucleotide probes, and are produced by photolithographic synthesis(Affymetrix) or by robotic printing. More recently, Maskless ArraySynthesis from NimbleGen Systems has combined flexibility with largenumbers of probes. Arrays can contain up to 390,000 spots, from a customarray design.

In pre-fabricated oligonucleotide microarrays or single-channelmicroarrays, the probes are designed to match the sequences of known orpredicted biomarkers. There are commercially available designs thatcover complete genomes from companies such as Affymetrix, or Agilent.These microarrays give estimations of the absolute value of geneexpression and therefore the comparison of two conditions requires theuse of two separate microarrays.

In some embodiments, the disclosure provides methods of screening forone or more biomarkers in a sample using PCR. The terms “PCR reaction”,“PCR amplification”, “PCR”, “pre-PCR”, “Q-PCR”, “real-time quantitativePCR” and “real-time quantitative RT-PCR” are used to signify use of anucleic acid amplification system, which multiplies the target nucleicacids being detected. Examples of such systems include the polymerasechain reaction (PCR) system and the ligase chain reaction (LCR) system.Other methods recently described and known to the person of skill in theart are the nucleic acid sequence based amplification and Q BetaReplicase systems. The products formed by said amplification reactionmay or may not be monitored in real time or only after the reaction asan end-point measurement.

In certain embodiments, the methods of the disclosure comprise screeningfor one or more biomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) in asample using real-time quantitative RT-PCR (qRT-PCR), qRT-PCR is amodification of polymerase chain reaction used to rapidly measure thequantity of a product of polymerase chain reaction. It is preferablydone in real-time, thus it is an indirect method for quantitativelymeasuring starting amounts of DNA, complementary DNA or ribonucleic acid(RNA). This is commonly used for the purpose of determining whether agenetic sequence is present or not, and if it is present the number ofcopies in the sample.

In some embodiments, the disclosure employs one of 3 methods of RT-PCR,or qRT-PCR. Like other forms of polymerase chain reaction, these methodsare used to amplify DNA samples, using thermal cycling and athermostable DNA polymerase. For example, the methods of the disclosureemploy RT-PCR using agarose gel electrophoresis, the use of SYBR Green,a double stranded DNA dye, or the used of one or more fluorescentreporter probes. The latter two of these are optionally analyzed inreal-time, constituting real-time polymerase chain reaction method.

In embodiments, the methods of the disclosure comprise detecting one ormore biomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) in a sampleusing RT-PCR, wherein the products of said RT-PCR are analyzed usingagarose gel electrophoresis. In exemplary embodiments, the unknownsample and a known sample are prepared with a known concentration of asimilarly sized section of target DNA for amplification. Both reactionsare run for the same length of time in identical conditions (preferablyusing the same primers, or at least primers of similar annealingtemperatures). Agarose gel electrophoresis is used to separate theproducts of the reaction from their original DNA and excess primers. Therelative quantities of the known and unknown samples are measured todetermine the presence or quantity of one or more biomarkers in theunknown sample. Accordingly, embodiments of the method are useful todetermine whether the probe target sequences of the one or morebiomarkers are present or not.

In other embodiments, the methods of the disclosure comprise detectingone or more biomarkers (e.g. 1, 2, 3, 4, 5, or more biomarkers) in asample using RT-PCR, wherein the products of said RT-PCR are analyzedusing SYBR green dye. In these embodiments, a DNA binding dye (SYBRgreen) binds all newly synthesized double stranded (ds)DNA, and anincrease in fluorescence intensity is measured, thus allowing initialconcentrations to be determined. A PCR reaction is prepared as usual,using primers directed to the one or more biomarkers disclosed herein,with the addition of fluorescent dsDNA dye. The PCR reaction is run, andthe levels of fluorescence are monitored. Comparing the level offluorescence in the sample to a reference standard sample or a standardcurve allows determination of the dsDNA concentration in the reaction,and thus a measure of the presence and quantity of starting material,comprising one or more biomarkers of the disclosure, in the sample.

In still other embodiments, the methods of the disclosure comprisedetecting one or more biomarkers (e.g. 1, 2, 3, 4, 5, or morebiomarkers) in a sample using RT-PCR, wherein the products of saidRT-PCR are analyzed using a fluorescent reporter probe. For example, asequence-specific nucleic acid based probe that recognizes the one ormore biomarkers of the disclosure is used to quantify the presence orlevels of said biomarkers in a sample. It is commonly carried out withDNA based probes with a fluorescent reporter and a quencher held inadjacent positions, so-called dual-labelled probes. The close proximityof the reporter to the quencher prevents its fluorescence; it is only onthe breakdown of the probe that the fluorescence is detected. Thisprocess depends on the 5′ to 3′ exonuclease activity of the polymeraseinvolved. The real-time quantitative PCR reaction is prepared with theaddition of the dual-labelled probe, thereby allowing accuratedetermination of the final, and so initial, quantities of one or morebiomarkers present in the sample.

In embodiments, the methods of the disclosure comprise one or moreprobes useful for detecting biomarkers (e.g. 1, 2, 3, 4, 5, or morebiomarkers) according to the methods disclosed herein. In someembodiments, the probes of the disclosure are useful for the detectionof a protein, phosphopeptide, nucleic acid and/or polypeptide molecule.In some embodiments, a probe of the disclosure is capable ofrecognizing, or detecting, one or more specific sequences of nucleicacid and/or polypeptide. In some embodiments, a probe of the disclosureis capable of recognizing a biomarker of the disclosure byhybridization. A nucleic acid according to the methods of the disclosurecomprises any nucleic acid, natural or synthetic, such as DNA, RNA,siRNA, LNA or PNA.

In some embodiments, a probe may be labeled, tagged, immobilized, orotherwise modified according to the requirements of the detection methodchosen. A label or a tag is an entity making it possible to identify acompound with which it is associated. It is within the scope of thepresent disclosure to employ probes that are labeled or tagged by anymeans known in the art such as but not limited to: radioactive labeling,fluorescent labeling and enzymatic labeling. Furthermore, the probe,labeled or not, may be immobilized to facilitate detection according tothe detection method of choice, and this may be accomplished accordingto the the particular detection method.

Another aspect of the present disclosure provides a kit, comprising: (a)a probe array for determining the presence or level of a dystoniabiomarker in a sample, the array comprising of a plurality of probesthat hybridizes to the biomarker or variants and mutations thereof thatare associated with dystonia and/or likelihood of developing dystonia;or (b) a kit for determining the presence of the biomarker in a sample,comprising the probe array of (a) and instructions for carrying out thedetermination of the presence of the biomarker in the sample. In someembodiments, the probe array of (a) further comprises a solid supportwith the plurality of probes attached thereto.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

The Examples that follow are illustrative of specific embodiments of theinvention and various uses thereof. They are set forth for explanatorypurposes only and are not to be taken as limiting the invention.

Examples Example 1 Development and Validation of Delta-E Torsin1aLocalization Assay

To develop an assay capable of detecting conditions that correct adystonia cellular phenotype, two human cell lines were generated thatstably expressed EGFP-tagged human wild-type Torsin 1A, or delta-ETorsin1a, from a single cDNA copy integrated at a defined genomic site(FIG. 1, panel 1).

To limit variation in expression levels associated with randomintegration or transient expression, single copies of either WT ordelta-E human TOR1A cDNAs with N-terminal EGFP fusions were inserted atthe FRT site in Flp-In™ T-Rex™ 293 cells (Thermo Fisher Scientific#R780-07) via flippase recombinase-mediated cassette exchange accordingto the manufacturer's recommended protocols. This system includes aninducible expression feature (TetON) that avoids selective pressure forpotential toxic effects of chronic expression of mutant proteins (FIG.1, panel 1).

Flp-In T-Rex 293 cells inducibly expressing either WT or delta-ETorsin1a were maintained in selective media [DMEM-HG (Thermo FisherScientific #11965)+1× GlutaMax (Thermo Fisher Scientific #35050-061)+75μg/mL hygromycin (Thermo Fisher Scientific #10687-010)+15 jpg/mLblasticidin S (Thermo Fisher Scientific #R210-01)+10% tetracyclinescreened FBS (Hyclone #SH30070.03T)+1%penicillin/streptomycin/amphotericin (Mediatech Inc. #30-004-Cl)]. Thecells were maintained at 37° C./5% CO2. All experiments were performedon cells with fewer than 5 passages. HEK 293T cells (ATCC #CRL-3216)were maintained in HEK-T media [DMEM (Thermo Fisher #11995)+10% FBS(Hyclone#SH30070.03)+1× GlutaMAX+1%penicillin/streptomycin/amphotericin] at 37° C./5% CO2. Human dermalfibroblast lines were maintained in FGM media [MEM (Thermo Fisher#11095-080)+15% FBS (Hyclone #SH30070.03)+1% Non-Essential Amino Acids(Lonza #13-114E)+1% penicillin/streptomycin/amphotericin] at 37° C./5%CO2.

This approach provides control over expression levels, avoiding the widevariation typically seen with random genomic integration or transientexpression approaches. Torsin1a expression was also controlled bytetracycline induction to avoid selective pressure modifying thecellular phenotypes. In this expression system, the WT and delta-ETorsin1a lines consistently and robustly reproduced their respectivesubcellular localization phenotypes (FIG. 1, panels a, b). The mutantphenotype was measured as the percentage of cells with one or more EGFPpuncta using automated high-content imaging analysis (see infra).

Upon establishing automated conditions to identify cells with thecharacteristic punctate EGFP signal, the predictive validity of theautomated readout in corroborating existing observations regardingTorsin1a biology was tested. First, compensation by the homologousprotein, Torsin1b, is hypothesized to underlie the selectivevulnerability of the brain where Torsin1b levels are low. (Jungwirth,M., et al., (2010) Hum. Mol. Genet. 19, 888-900.) Consistent with thismodel, knockdown of Torsin1b significantly increased Torsin1amis-localization in both the WT and delta-E cell lines (FIG. 1, panelc). % Selected Cells refers to percent of cells that have one or morepuncta of EGFP signal as determined by automated image analysis. N=16independent wells treated with non-silencing siRNA control and 32independent wells treated with TOR1B siRNAs. *, p<0.05; ***, p<0.0005 byunpaired t test.

Second, steady-state protein levels of Torsin1a have also beenhypothesized to contribute to DYT1 dystonia pathogenesis. (Jungwirth,M., et al., (2010) Hum. Mol. Genet. 19, 888-900; Kim, C. E., et al.,(2010) Proc. Natl. Acad. Sci. U.S.A. 107, 9861-6; Hewett, J. W. et al.,(2007) Proc. Natl. Acad. Sci. U.S.A. 104, 7271-6.) That model supportedby observations in transgenic mouse models. (Goodchild, R. E. & Dauer,W. T. (2004) Proc. Natl. Acad. Sci. U.S.A 101, 847-52.) Indeed, we foundthat inhibiting protein clearance with the proteasome inhibitor MG132(MG132, 10 μM) significantly increased the percentage of cells withpunctate EGFP-Torsin1a signal in both WT and delta-E cell lines (FIG. 1,panel d).

Lastly, expression of delta-E Torsin1a has been associated withincreased ER stress and activation of the unfolded protein response(UPR). (Chen, P. et al. (2010) Hum. Mol. Genet. 19, 3502-15; Bragg, D.C., et al., (2011) Neurobiol. Div. 42, 136-47; Nery, F. C. et al. (2011)Nat. Commun. 2, 393; Cao, S. et al. (2010) Dis. Model. Mech. 3, 386-96.)In addition, the chemical chaperone phenylbutyric acid (PBA) hasrecently been shown to reduce signs of ER stress in DYT1 patient-derivedfibroblasts. (Barrows, N. J., et al., (2010) J. Biomol. Screen 15,735-47.) We also found that PBA (20 mM) ameliorated the burden of cellswith punctate pathology in our assay cell lines (FIG. 1, panel d).

Importantly DMSO vehicle had no effect on the number of cells one ormore puncta of EGFP signal. (n=4 independent DMSO-treated wells and 8independent wells each for MG132 and PBA treatments. ***, p<0.0005 byunpaired t test.)

Thus, these observations support the utility of the phenotypic screen asa tool to identify factors that modulate delta-Torsin1amis-localization, and by extension dystonia phenotypes.

Image Analysis:

Development and validation of the high-content image analysis and assayperformance under high-throughput screening conditions is presented inFIG. 9. The image analysis algorithm was developed using the CellomicsArrayScan V CompartmentAnalysisV2 protocol. Hoechst's nuclear staining(Ch1: excitation 350 nm; emission 461 nm) was used for focusing and forcell identification by nuclear size, shape, and intensity. Automatedexposure times for each plate were determined by setting a targetsaturation of 25% in channel 1 in wells containing tetracycline-induceddelta-E cells. Detected cells were gated according to nuclear area,length-to-width ratio, and perimeter-to-area ratio. For the purpose ofanalysis, cell bodies were defined by a radius of 10 μm from the outeredge of the defined nucleus. Overlapping cells were automaticallysegmented according to the Cellomics ArrayScanV CompartmentAnalysisV2object segmentation feature. EGFP fluorescence (Ch2: excitation 488 am;emission 509 nm) was used for puncta detection. Automated exposure timesfor each plate were determined by setting a target saturation of 35% inchannel 2 in wells containing induced delta-E cells. Torsin1a punctawere identified using the spot detection feature of the CompartmentalAnalysisV2 protocol. The percentage of cells containing one or morepuncta was used as the primary metric in this assay. As a technicalcontrol for the specificity of the signal, we confirmed that RNAitargeting Torsin1a eliminated inclusions (FIG. 9, panel e).

Messenger RNA Expression Profiling of Assay Cell Line

The gene expression profile of cells inducibly expressing delta-ETorsin1a was evaluated to identify mRNAs that are differentiallyexpressed in the DYT1 model system.

Flp-In T-Rex 293 cells inducibly expressing delta-E Torsin1a were seededat 2×106 cells per 10 cm2 dish. Cells were treated with tetracycline (5μg/mL, Sigma #87128) or PBS (uninduced control) 24 h after initialplating. Cells were harvested 72 h after tetracycline induction. RNA wasextracted using the Rneasy RNA extraction kit (Qiagen #74104) as permanufacturer's protocol. Microarray expression profiling was performedat the Duke Center for Genomic and Computational Biology (Durham, N.C.)using standard protocols and the GeneChip4® Human Genome U133 Plus 2.0Array (Affymetrix). The Affymetrix Gene Chip microarray data underwentstrict quality control processing using the simpleaffy package inBioconductor. Log-scale Robust Multiarray Analysis (RMA) from the affypackage in Bioconductor was used for normalization to eliminatesystematic differences across the arrays. The mass algorithm from theaffy package was used to make present/absent calls. Bioinformaticsanalysis of RNAi screen hits was performed exclusively on probesetsfound to be present.

Differential gene expression data from microarray analysis shows thatexpression of 4 genes are up-regulated in response to EGFP-delta-ETorsin1a induction for 72 hours in our assay cell lines: HERPUD1,HSPA5(BiP), RAP1A, and MAP2K5 (Table 1).

TABLE 1 mRNAs differentially expressed in delta-E Torsin1a expressingcells geneName Tet.plus.mean Tet.minus.mean FoldChange p. value p. adjRAP1A 10.65499601 6.25273683 4.40225918 4.26E−014 1.65E−009 RAP1A10.76761501 5.536435372 5.231179638 6.02E−014 1.65E−009 MAP2K56.669384725 5.459748948 1.209635777 8.34E−008 0.001519571 HERPUD112.03560636 11.40806375 0.627542606 1.58E−006 0.021536699 HSPA512.27441717 11.63766644 0.636750727 1.99E−006 0.021803043 HSPA58.553374401 7.893659664 0.659714737 2.78E−006 0.02531687

Example 2: Whole Genome siRNA Screen to Correct Delta-E Torsin1aMis-Localization

We first performed a pilot screen targeting 960 genes from the QiagenWhole Genome siRNA Knock-Down Library 1.0 to determine the robustness ofthe assay system under automated high-content imaging, high-throughputscreening conditions, and to establish that gene knockdown using siRNAcould normalize delta-E Torsin1a distribution (FIG. 1, panels e-g; seealso, FIG. 9, panel c). We thereby identified suitable screeningconditions and a positive control siRNA pair (e.g., FIG. 1, panel g). WTand delta-E treated with non-silencing siRNA control or with positivecontrol siRNA showed the ability to rescue the delta-E Torsin1amis-localization phenotype (FIG. 1, panels e-g.) The reproducibility ofthe assay was established in multiple runs (FIG. 1, panel h).

The genome-wide RNAi screen on Flp-In T-Rex 293 cells induciblyexpressing either WT or delta-E Torsin1a was performed at the DukeUniversity RNAi screening facility (Durham, N.C.) by using the Qiagengenomic siRNA library v 1.0 consisting of four distinct siRNAs (A, B, C,and D) targeting 22,909 known and putative human genes as described in27. Pairs of siRNAs (either AB or CD) in 5 μL of water (1 pmol/well)were added to clear-bottom 384-well plates (Corning #3712). Whereappropriate, non-silencing siRNA (Qiagen #SI03650325) or TOR1B siRNAs(Qiagen #s SI00749574, SI04135404, SI04221728, and SI04270672) wereadded instead of library siRNAs. OptiMEM (Thermo Fisher #11058-021)+0.5%Lipofectamine RNAiMAX (Thermo Fisher #13778150) (10 μL/well) was added20 min prior to cell plating. Flp-In T-Rex 293 WT or delta-E Torsin1acells were plated at 3,000 cells/well in 50 μL assay media [DMEM (ThermoFisher #11965)+1% tetracycline-screened FBS (Hyclone #SH30070.03T)+1×GlutaMAX (Thermo Fisher #35050-061)+1%penicillin/streptomycin/amphotericin (Corning#30-004-Cl)], and incubatedat 37° C./5% CO2 overnight. The following day, 15 μL assay media+26.7μg/mL tetracycline (final tetracycline concentration: 5 μg/mL) was addedto all wells except the un-induced control. Seventy-two hours afterinduction, cells were fixed with 4% PFA (Sigma #P6148) in PBS,permeabilized in PBS+0.5% Triton X-100 (Sigma #T8787), and stained withHoechst 33342 nuclear dye (Sigma #B2261, 13.3 μg/mL in PBS). Plates werethen sealed and imaged on a Cellomics ArrayScan automated high-contentimaging system.

After identifying suitable screening conditions described above, a wholegenome screen (WGS) targeting 22,909 human genes was performed (QiagenWhole Genome siRNA Knock-Down Library 1.0). Four siRNAs targeting eachgene were tested in a paired-screen design of two independent pools oftwo siRNAs each (FIG. 1, panel h). (See Panda, D. et al. (2011) Proc.Natl. Acad. Sci. U.S.A 108, 19036-41; Barrows, N. J. et al., (2014)Methods Mol. Biol. 1138, 285-99; Harding, H. P. et al., (2003) Mol. Cell11, 619-33.) Primary hits were defined as those genes whose knockdownimproved delta-E Torsin1a localization by at least 3 standard deviations(from the mean of the non-silencing control siRNA (NSC)) and the effectwas concordant, e.g. present in both of the two unique siRNA pools (FIG.1, panel i). Hits that were cytotoxic, significantly decreasedEGFP-Torsin1a expression, or were not expressed in the assay cell line(as determined by microarray analysis) were discarded. The primaryimages were then examined by blinded scorers and ranked to identifythose hits that resulted in typical wild-type cellular morphology andTorsin1a distribution. The resulting group was used for pathway analysisand consisted of 93 high-stringency and high-quality hits (FIG. 1, panelj; Table 2).

TABLE 2 Gene hits used for pathway analysis HUGO Entrez Gene Symbol GeneID AASS 10157 ACOT13 55856 AGGF1 55109 ANXA3 306 ARHGEF11 9826 ATG9A79065 ATP13A1 57130 ATP5A1 498 BDP1 55814 BOP1 23246 C16orf58 64755C5orf44 80006 CCDC86 79080 CCZ1 51622 CDCA4 55038 CEBPD 1052 CEP78 84131CLEC4A 50856 CNOT6L 246175 COASY 80347 CTRB1 1504 DDX24 57062 DHX15 1665DIXDC1 85458 DLL3 10683 DNM1 1759 DUSP5 1847 EPDR1 54749 ERMP1 79956ERO1LB 56605 EXOSC1 51013 FAM92B 339145 FLRT2 23768 GOLGA1 2800 GP1BB2812 HERC2P9 440248 HHEX 3087 HIGD2B 123346 HNRPULI 11100 JPH1 56704K1AA1257 57501 KMT2A 4297 LOC285033 285033 LOC286186 286186 LOC441204442519 MAML2 84441 MERTK 10461 MRC2 9902 NGLY1 55768 NTPCR 84284 P4HTM54681 PBX2 5089 PCDHGB3 56102 PHACTR3 116154 POLR2E 5434 POLR2K 5440PPP1R16A 84988 PRRG1 5638 PSMD4 5710 PTF1A 256297 PVT1 5820 RBM3 5935RPAP3 79657 RPL10A 4736 RPL10L 140801 RPL11 6135 RPL12 6136 RPL39 6170RPL5 6125 RPL6 6128 RPUSD4 84881 SCD 6319 SH2D3C 10044 SPANXC 728712SPDYE4 388333 SPPL2A 84888 SPTLC1 10558 SRGAP3 9901 SRP19 6728 SRP686730 SRPR 6734 SSBP2 23635 TAF15 8148 TGFB1 7040 TMEM258 746 TRAPPC327095 UBE21 7329 UGT8 7368 UNG 7374 WBSCR22 114049 W1BG 84305 XAGE1D9503 Y1PF3 25844

Example 3: Hit Validation and Bioinformatics Analysis

To cross-validate WGS hits in a system that did not rely upon exogenousexpression of Torsin1a or a fusion protein, four of the highest-qualityhits were tested in an orthogonal counter screen using patient-derivedfibroblasts. We tested each shRNA for its ability to rescue a previouslydescribed defect in luciferase secretion. Chen, P. et al. (2010) Hum.Mol. Genet. 19, 3502-15; Cao, S. et al. (2010) Dis. Model. Mech. 3,386-96; Barrows, N. J., et al., (2010)J. Biomol. Screen. 15, 735-47.)DYT1 patient data were obtained from 4 independent patient-derived linesand WT data (black bar) from 3 normal healthy control lines. Each linewas tested in 5 independent experiments that included 4 technicalreplicates each (with the exception of SCD data from 3 independentexperiments). Three of the four hits tested also improved thisDYT1-related phenotype (FIG. 1, panel k). Bioinformatic analysis wasthen conducted, which identified eleven significantly over-representedsignaling pathways (Table 3).

TABLE 3 Bioinformatic analysis of whole genome RNAi screen hits.Ingenuity Canonical Pathways p-value 1 EIF2 Signaling (6) 0.000098 2Assembly of RNA Polymerase II Complex (3) 0.0011 3 Cell Cycle: G1/SCheckpoint Regulation (3) 0.0019 4 Glucocorticoid Receptor Signaling (5)0.0041 5 Nucleotide Excision Repair Pathway (2) 0.0087 6 Notch Signaling(2) 0.010 7 Coenzyme A Biosynthesis (1) 0.012 8 Estrogen ReceptorSignaling (3) 0.015 9 Lysine Degradation II (1) 0.020 10 CeramideBiosynthesis (1) 0.024 11 Oleate Biosynthesis II (Animals) (1) 0.048

The most highly enriched pathway among WGS hits—as well as among hitsderived from an additional, less stringent WGS analysis (22SD effectsize), was eukaryotic initiation factor 2a (eIF2-alpha) signaling, alsoknown as the integrated stress response (ISR) (FIG. 2a ). (Sidrauski, C.et al., (2013) Elife 2, e00498; Hinnebusch, A. G. & Lorsch, J. R.,(2012) Cold Spring Harb. Perspect. Biol. 4.) Briefly, in the integratedstress response, eIF2-alpha, the rate-limiting regulatory subunit of theeIF2 complex which mediates the binding of methionyl-tRNA to theribosome to begin protein translation (Vattem, K. M. & Wek, R. C.,(2004) Proc. Natl. Acad. Sci. U.S.A 101, 11269-74), is phosphorylated byfour upstream stress-sensitive kinases. Phosphorylation of eIF2-alphahas two main effects: a general decrease in the rate of proteintranslation and an increase in the translation of a subset oftranscripts containing upstream open reading frames (uORFs). (Jackson,R. J., et al., (2010) Nat. Rev. Mol. Cell Biol. 11, 113-27; Harding, H.P. et al., (2000) Mol. Cell 6, 1099-108.) Principal among thetranscripts whose translation is upregulated is ATF4, a transcriptionfactor which stimulates the expression of stress response proteins.(Jackson, R. J., et al., (2010) Nat. Rev. Mol. Cell Biol. 11, 113-27;Fullwood, M. J., et al., (2012) Prog. Mol. Biol. Transl. Sci. 106,75-106.) Eukaryotic initiation factor 2a signaling also leads to theexpression of the protein phosphatase 1 regulatory subunits CreP andGADD34, which dephosphorylate eIF2-alpha and terminate ISR activation.(Boyce, M. et al., (2005) Science 307, 935-9.)

Example 4: Pharmacological Characterization of the Effects of eIF2-AlphaSignaling on Torsin1A Localization

Although the screen implicated eIF2-alpha signaling, the hits thatyielded the bioinformatics result (FIG. 2, panel b) did not reveal thedirectionality of the signaling change normalizing delta-E Torsin1alocalization. However, knockdown of each of the four eIF2-alpha kinasessignificantly worsened delta-E Torsin1a localization (FIG. 2, panels a,b) indicating that decreased eIF2-alpha signaling has a deleteriouseffect on delta-E Torsin1a localization. These results also suggest amechanism in which promoting eIF2-alpha pathway signaling, e.g.suppression of eIF2-alpha activity, would be beneficial.

In order to test this model directly, the delta-E Torsin1a cell line wastreated with compounds targeting the eIF2-alpha signaling pathway (FIG.2). Flp-In T-REx 293 cells inducibly expressing either WT or delta-ETorsin1a were plated in clear-bottom 384-well plates (Corning #3712) at3,000 cells/well in 30 ipL assay media [DMEM (Thermo Fisher #11965)+1%tetracycline-free FBS (Hyclone #SH30070.03T)+1× GlutaMAX (Thermo Fisher#35050-061)+1% penicillin/streptomycin/amphotericin (Corning#30-004-Cl)], and incubated at 37° C./5% CO² overnight. The followingday, serial drug dilutions were prepared at 2× final concentration inassay media containing 10 μg/mL tetracycline and 30 μL of thedrug/tetracycline mixture was added to the appropriate wells, whilecontrol wells received 30 μL of assay media alone or assay media+10 g/mLtetracycline (final tetracycline concentration for all wells exceptnegative controls: 5 μg/mL). Cells were then incubated at 37° C./5% CO²for 48 hours and fixed, permeabilized, Hoechst stained, and imaged asdescribed above. The range of the Torsin1a localization effect wasnormalized to the percentage of cells with puncta in vehicle-treateddelta-E cell line as the maximum and that in WT cell line as the minimum(FIG. 2, panels c-h). Cell count and EGFP-Torsin1a expression werenormalized to their respective values in the vehicle-treated delta-Ecell line as the maximum. All dose response data are the average of 4independent experiments per dose. Untreated control data used fornormalization are the average of 24 independent experiments.

Salubrinal (R&D Systems #2347), a drug that prolongs ISR activation bypromoting eIF2-alpha phosphorylation (Tsaytler, P., et al., (2011)Science 332, 91-4), caused a robust dose-dependent normalization ofdelta-E Torsin1a localization, without modifying steady-stateEGFP-Torsin1a levels or significant toxicity or obvious alterations ofcellular morphology at effective concentrations (EC50=2.12 μM) (FIG. 2,panel c). As a control, we confirmed that treatment with vehicle (DMSO)at concentrations present in these experiments had no effect on the rateof delta-E or WT Torsin1a inclusions or cell number (FIG. 2, panel d).In addition, none of the compounds tested significantly alteredsteady-state levels of EGFP-Torsin1a (FIG. 2, grey triangles in allpanels).

Another eIF2-alpha phosphatase inhibitor, guanabenz, had no effect ondelta-E Torsin1a localization (FIG. 2, panel e). Guanabenz, however, isa more selective inhibitor of GADD34-containing eIF2-alpha phosphatasecomplexes (Stockwell, S. R. et al., (2012) PloS One 7, e28568),suggesting that activity of both GADD34- and CreP-containing PPIcomplexes may need to be silenced in order to normalize delta-E Torsin1alocalization. Consistent with this possibility, neither GADD34 nor CrePwas identified as a hit in the RNAi screen (FIG. 2, panel b).

We next examined the efficacy of a direct activator of the eIF2-alphakinase PERK, CCT02031238. CCT020312 also caused a dose-dependentreduction in the number of cells with Torsin1a inclusions. However,CCT020312 treatment was highly toxic at effective concentrations, asindicated by both cell count and cellular morphology. Thus, collectivelythese experiments suggest that positive modulation—as opposed to directactivation—of eIF2-alpha signaling may be more easily tolerated andtherefore more likely to be of therapeutic value.

Upon finding that prolonged signaling through eIF2-alpha phosphorylationnormalizes delta-E Torsin1a distribution (e.g., FIG. 2, panel c), it wasthen determined whether preventing such signaling would alter the normaldistribution of Torsin1a. To do this, the compound ISRIB, which preventsthe downstream signaling consequences of eIF2-alpha phosphorylation,(Hinnebusch, A. G. & Lorsch, J. R., (2012) Cold Spring Harb. Perspect.Biol. 4; Patil, C. & Walter, P., (2001) Curr. Opin. Cell Biol. 13,349-55), was tested on the WT Torsin1a-expressing assay cell line. ISRIBcaused a dose-dependent increase in cells with punctate Torsin1alocalization without effects on Torsin1a expression levels (FIG. 2,panel f). These findings indicate a dynamic role for the ISR pathway inbi-directionally regulating Torsin1a localization.

Finally, compounds were tested targeting two other pathways enrichedamong the WGS hits, notch signaling and glucocorticoid signaling, andanother ER stress response pathway. Treatment with the γ-secretaseinhibitor, DAPT, which blocks notch signaling, had no effect on delta-ETorsin1a localization (FIG. 2, panel g), nor did the anticonvulsant,valproic acid, which, among other activities, activates notch signaling.Similarly, neither the glucocorticoid receptor agonist dexamethasone(FIG. 2, panel h), or a small panel of additional compounds modulatingglucocorticoidimineralocorticoid receptor signaling significantlymodified delta-E Torsin1a localization. Lastly, because PERK-mediatedeIF2-alpha signaling is one of three branches of the unfolded proteinresponse (Dang, M. T. et al., (2005) Exp. Neurol. 196, 452-63),modulators that are available for another branch, which requires ATF6signaling, were tested. We found that compounds targeting this branchprimarily caused cytotoxicity. Furthermore, knockdown of ATF6 did notsignificantly normalize or worsen delta-E Torsin1a localization in theWGS screen.

Together, these results identify augmentation of signaling through theeIF2-alpha pathway as a specific, efficacious and non-toxic target tonormalize delta-E Torsin1a mis-localization.

Example 5: Expression of ATF4 is Sufficient to Normalize Delta-ETorsin1a Localization

The mechanisms downstream of eIF2-alpha phosphorylation that might beinvolved in mediating the normalizing effects of ISR signaling ondelta-E Torsin1a localization was investigated. Phosphorylation ofeIF2-alpha has two downstream consequences: a decrease in the generalrate of protein translation and an increase in translation of a subsetof transcripts containing uORFs, the most well-characterized of which isthe transcription factor ATF4 (FIG. 2a ). (Jackson, R. J., et al.,(2010) Nat. Rev. Mol. Cell Biol. 11, 113-27.) Therefore, to determine ifincreased ATF4 expression alone was sufficient to normalize delta-ETorsin1a localization, we overexpressed ATF4 in the delta-E Torsin1aassay cell line.

Flp-In T-REx 293 cells inducibly expressing either WT or delta-ETorsin1a were plated as described above. The following day, cells weretransfected with empty vector (pBluescript) or pRK/FLAG-ATF4 usingLipofectamine2000 (Thermo Fisher #11668) and Opti-MEM (Thermo Fisher#11058) according to the manufacturer's instructions (56 ng DNA/well, 10μL total volume/well). 4 hours later, 20 L of assay media+15 μg/mLtetracycline was added to each well (5 μg/mL final concentration), andcells were incubated at 37° C./5% CO² for 48 hours. Cells were thenfixed, permeabilized, Hoechst stained, and imaged as described above.After initial imaging, cells were stained for FLAG-ATF4 as follows: 50μL blocking solution [10% normal goat serum (Thermo Fisher #16210-064)in PBS] was added to each well and the cells were incubated for 20′ atRT. Excess blocking solution was then aspirated and 50 μL primaryantibody solution [mouse anti-FLAG Mj2 (Sigma #F3165), 1:1000 inblocking solution] was added to each well for 30′ at RT. Cells were thenwashed twice with PBS and 50 μL secondary antibody solution[Alexa594-conjugated goat anti-mouse (Thermo Fisher #A-11005), 1:1000 inblocking solution] was added to each well for 30′ at RT. Lastly, plateswere washed 4 times with PBS, sealed, and re-imaged.

Representative images are presented in FIG. 3, panel (a). Quantitateddata are presented in FIG. 3, panel (b). Quantitation range of theTorsin1a localization effect was normalized to the percentage of cellswith puncta in vector control-transfected delta-E cell line as themaximum and that in WT cell line as the minimum. N=16 independent wellsfor each condition, and an additional 24 untransfected control wellsused for normalization.

ATF4 overexpression significantly improved delta-E Torsin1alocalization, with negligible effects on WT Torsin1a localization (FIG.3a ). Moreover, the magnitude of the effect was similar betweensalubrinal-treated and ATF4-expressing cells (FIGS. 2c and 3b ;salubrinal efficacy=66.4%, ATF4 efficacy=57.0%).

Example 6: Enhancing eIF2-Alpha Signalling Improves Neonatal Survival ofHomozygous DYT1 Knock-in Mice

The findings above support a role for the eIF2-alpha signaling pathwayin normalizing in vitro cellular phenotypes related to the DYT1genotype. Next, the effect of targeting eIF2-alpha signaling on thedeleterious consequences of the DYT1 TorsinA mutation was determined invivo. Etiological mouse models of DYT1 dystonia have not had robustdystonic phenotypes. (Tanabe, L. M., et al., (2012) PloS One 7, e32245;Camargos, S. et al., (2008) Lancet. Neurol. 7, 207-15; Seibler, P. etal. (2008) Lancet. Neurol. 7, 380-1.) Nonetheless, another deleteriousgenotype-dependent effect of the mutation on the animal-neonatallethality of homozygous delGAG knock-in mice—facilitated interrogationof the impact of eIF2-alpha targeting in vivo (FIG. 4a ). (Seibler, P.et al. (2008) Lancet. Neurol. 7, 380-1; Goodchild, R. E., et al, (2005)Neuron 48, 923-32.)

Breeding and Viability Determination:

Pairs of heterozygous delta-E Torsin1a knock-in breeders (courtesy ofDr. W. Dauer, University of Michigan) were randomly assigned to vehicleor salubrinal treatment groups. Starting 10 days after breeding cageswere established, each female mouse was given a daily subcutaneousinjection of vehicle or salubrinal at approximately 6 p.m. Cages werechecked for new pups three times per day: morning (˜8 a.m.), earlyafternoon (˜2 p.m.), and during injections (˜6 μm.). In order tominimize stress and the possibility of litter abandonment, mice withnewborn pups were not given injections. Newborn pups were identified bymarking limbs with a permanent laboratory marker, and tail samples weretaken for genotyping at P0. At approximately P0.5, cages were checkedagain, and the status of each pup was recorded. If pups were firstobserved at the 8 am check, the 2 μm check was considered P0.5. If pupswere first observed at the 2 μm or 6 μm checks, an additional check wasperformed at approximately midnight, and considered P0.5. Mortality wasalso tracked the following day, after which time identifying marks werepredominately washed off or obscured by fur. Approximately 8 days afterbirth all remaining pups were sacrificed, and 10 days after birth dailyinjections resumed for the subsequent litter. All genotyping and,whenever possible, all pup identification and mortality checks wereperformed blinded to treatment group.

Drug Administration:

Salubrinal (R&D Systems #2347) stocks were prepared at 26 mM (10× finalconcentration) in DMSO and stored in single-use aliquots at −80° C. Eachday immediately before injections, a single salubrinal aliquot wasthawed to room temperature and diluted 1:10 in injection vehicle (finalvehicle composition: 1×PBS/10%/DMSO/0.1% BSA). Empty injection vehiclewas stored at room temperature in single-use aliquots. Mice were given asingle subcutaneous injection of 3.3 mg/kg salubrinal (approximately 80μL for a 30 g mouse) or an equivalent volume of empty injection vehicle.

N=29 vehicle- and 28 salubrinal-treated homozygous pups and 65 vehicle-and 91 salubrinal-treated pooled heterozygous and wild-type pups. Nosignificant drug effect on mortality was observed for either wild-typeor heterozygous genotype, so results were combined to simplifypresentation (FIG. 4, panel b).

The eIF2-alpha phosphatase inhibitor salubrinal was administered dailyto pregnant dams (in Tor1adeIGAG/+× Tor1adeIGAG/+breedings) fromapproximately embryonic day 10 through delivery and tested its effectson neonatal survival (FIG. 4, panel a). In comparison to litters fromvehicle-treated dams, salubrinal dramatically reduced the mortality ofdelta-E/delta-E pups (FIG. 4, panel b). Additionally, there was noevidence that salubrinal had a non-specific mechanism in improvingneonatal survival, as no mortality differences were observed betweentreatment and control groups in the remaining pups of either wildtype orheterozygote genotypes (FIG. 4, panel b). Although in this study designthere was no postnatal salubrinal treatment except possibly throughlacteal transfer (though homozygous delta-E/delta-E pups are known tonot nurse), we anecdotally note that two salubrinal-treated homozygousdelta-E Torsin1a pups survived through the second postnatal day. Novehicle-treated homozygous pups survived through even the firstpostnatal day.

Example 7: Stress-Induced eIF2-Alpha Signaling is Impaired in Cells fromHuman DYT1 Patients

As described above, pharmacologically enhancing eIF2-alpha signaling cantreat in vitro and in vivo DYT1 genotype-related dystonia phenotypes.However, a key remaining question was whether the eIF2-alpha signalingpathway was disrupted in DYT1 dystonia. To address this, we examined theintegrity of the eIF2-alpha signaling pathway in fibroblasts derivedfrom human DYT1 patients. We measured the well-known stress-inducedincrease in ATF4 expression as a readout of pathway activation.

Normal control and DYT1 patient human dermal fibroblasts were plated in6-well plates at 25,000 cells/well in FGM media (2 mL/well) andincubated 37° C./5% CO2 for 4 days. Media was replenished with FGM (1mL/well) and incubated at 37° C./5% CO2 overnight. Opti-MEM (1 mL/well)containing 2 μg/mL thapsigargin (Santa Cruz Biotechnology #sc-24017) wasadded to cells and incubated as indicated (final concentration: 1μg/mL).

Cells were harvested in RIPA buffer [150 mM NaCl/50 mM NaH2PO4/2 mMEDTA/l % Triton X-100/0.5% SDS/0.5% deoxycholic acid/50 mM NaF/10 mMNa4P207/1 mM Na3VO4/1× phosphatase inhibitor cocktail (Sigma #P5726)/1×cOmplete Mini EDTA-free protease inhibitor cocktail (Roche#04693159001)]. Total protein concentrations were assessed by BCA assay(Thermo Fisher Scientific #23225). Proteins were resolved on 4-15% TOXgels (Bio-Rad #5671085), transferred to nitrocelulose membrane, blockedin TBS-T+5% non-fat dry milk, and probed as indicated. (Primaryantibodies employed in the examples of the disclosure are presented inTable 4.) ATF4 expression was normalized to 1-actin expression. N=3independent control cells lines and 4 independent DYT1 cell lines, 3replicates each.

TABLE 4 Primary antibodies used in the Examples. Reactivity SupplierCat. # Dilution β-Actín Millipore MAB1501 1:1,000 FLAG Sigma F31651:1,000 ATF4 Santa Cruz Biotechnology, Inc. SC-200 1:500 CrePProteintech 14634-1-AP 1:500 GAPDH Abcam ab9485 1:1,000

Representative images and quantitation are presented in FIG. 5, panelsa, b. Strikingly, we found that ATF4 upregulation in response to the ERstressor thapsigargin was impaired in DYT1 patient-derived fibroblastsrelative to fibroblasts from patients without disease (FIG. 5). Thisresult provides evidence that eIF2-alpha signaling is disrupted in DYT1dystonia.

Example 8: Identification of a Loss-of-Function ATF4 Mutation inSporadic Dystonia Patients

Interestingly, another rare inherited dystonia supports our findingsimplicating deficient eIF2-alpha signaling in DYT1 dystoniapathogenesis. Mutations in the PRKRA gene, which encodes an upstreameIF2-alpha kinase activator (FIG. 2a ), cause DYT1 6 dystonia. (Zech,M., et al., (2015) Mov. Disord 30, 878-9; Vaughn, L. S. et al., (2015)J. Biol. Chem. 290, 22543-57; Lassot, I. et al., (2001) Mol. Cell. Biol.21, 2192-202.) Functionally, the most common PRKRA mutation impairs theeIF2-alpha signaling pathway by delaying and reducing eIF2-alphaphosphorylation. (Frank, C. L., (2010) J. Biol. Chem. 285, 33324-33337.)Considering the intersection of this known cause of DYT16 with ourfindings newly implicating DYT1 dystonia in the eIF2-alpha pathway, itwas hypothesized that eIF2-alpha pathway dysfunction might alsocontribute to instances of non-familial sporadic dystonia.

To investigate this possibility, exomic sequences from 20 patientspresenting with sporadic dystonia were examined. (FIG. 5, panel c). 20subjects with sporadic dystonia were exome sequenced. Results werecompared to sequence from 573 control samples that were sequenced aspart of other studies at Duke University. All studies were reviewed andapproved by the Duke University Medical Center IRB, and all subjectsgave written informed consent. Dystonia subjects were recruited at theMovement Disorders Center at Duke University Medical Center, Durham,N.C.

We collected 20 unrelated patients diagnosed with adult onset, sporadicdystonia (3 men and 17 women) with a mean age of dystonia onset of47.4+/−9.59 years. A diagnostic workup was conducted by a MovementDisorders specialist to confirm the symptoms of dystonia with muscleinvolvement classified as focal, segmental, multifocal, or generalized.Only presumptive primary cases were recruited. Secondary dystoniasassociated with conditions such as Parkinson's disease or otherneurodegenerative diseases were excluded. Cases suggestive of Mendelianinheritance were also excluded. A complete family and medical historywas collected including common toxic exposures and medicalcomorbidities. Control samples were sequenced as part of other studiesat Duke University Medical Center and were not enriched for (but notspecifically screened for) dystonia or other neurological disorders.

Sequencing of DNA was performed at Duke University. Samples were exomesequenced using the Agilent All Exon 37 MB or 50 MB kit using IlluminaGAIIx or HiSeq 2000 or 2500 sequencers according to standard protocols.All samples were processed using the same methods, as follows. TheIllumina lane-level fastq files were aligned to the Human ReferenceGenome (NCBI Build 37) using the Burrows-Wheeler Alignment Tool (BWA).We then used Picard software (picard.sourceforge.net) to removeduplicate reads and process these lane-level SAM files, resulting in asample-level BAM file that is used for variant calling. GATK was used torecalibrate base quality scores, realign around indels, and callvariants. Variants were required to have a quality score (QUAL) of atleast 20, a genotype quality (GQ) score of at least 20, at least 10×coverage, a quality by depth (QD) score of at least 2 and a mappingquality (MQ) score of at least 40. Indels were required to have amaximum strand bias (FS) of 200 and a minimum read position rank sum(RPRS) of −20. SNVs were restricted according to VQSR tranche(calculated using the known SNV sites from HapMap v3.3, dbSNP, and theOmni chip array from the 1000 Genomes Project): the cutoffs were atranche of 99.9%. Variants were excluded if marked by EVS as beingfailures. Variants were annotated to Ensembl 73 using SnpEff. Onlygenetically European ethnicity samples were included in the analysis.Samples were screened with KING to remove second-degree or higherrelatives; samples with incorrect sexes according to X:Y coverage ratioswere removed, as were contaminated samples according to VerifyBamID. Weused Analysis Tools for Annotated Variants (igm.cumc.columbia.edu) toidentify coding variants that were found in at least 2 cases and 0controls. The presence of mutations in ATF4 causing the P46L amino acidsubstitution were confirmed by Sanger sequencing.

The results of exome analysis are presented in FIG. 5, panel c. In theseexomes, an analysis was performed to identify unique missense variants(relative to 573 controls) that were present in at least two cases.Genome-wide (i.e. agnostic to hypothesized mechanism), this analysisidentified only 14 rare missense coding mutations that met thiscriterion (FIG. 5, panel c). Notably, one of these mutations was in thegene for the phospho-eIF2-alpha effector ATF4 (n.22:39917587C/T,rs111719524, p.P46L).

To evaluate the functional significance of this mutation, we tested thetranscriptional activation activity of P46L ATF4 using a luciferasereporter driven by the ATF4-sensitive amino acid response element (AARE)(FIG. 5, panel d). As controls, we tested the activity of similarlyexpressed WT protein and a common ATF4 polymorphism, Q22P, which ispresent in approximately one third of the human population(exac.broadinstitute.org).

Constructs and Mutagenesis:

P46L and Q22P mutations were introduced into pRK/FLAG-ATF4 using aQuikChange Lightning site-directed mutagenesis kit (Agilent #210518)according to the manufacturer's instructions. Amino acid responseelement-sensitive Renillia luciferase (AARE-Rluc) was obtained fromSwitchGear Genomics (#S900027). Constitutively expressed Cyperidinaluciferase was obtained from New England Biolabs (pCMV-Cluc 2; #N0321S).pAARE-Rluc, pCMV-Cluc 2, and wildtype and mutant ATF4 sequences wereconfirmed by Sanger sequencing.

AARE Reporter Assay:

HEK293T cells were plated in 6-well plates (Corning #3506) at 500,000cells per well in 2 mL HEK-T media [DMEM (Thermo Fisher#11995)+10%0/FBS+1× GlutaMAX+1× penicillin/streptomycin/amphotericin]and incubated at 37° C./5% CO2 overnight. The following day, cells weretransfected with pAARE-Rhuc, pCMV-Cluc2, and empty vector or WT/mutantATF4 as appropriate, using Lipofectamine2000 and OptiMEM according tothe manufacturer's instructions (2.5 μg total DNA/well, 500 μL totalvolume/well). After 4 hours, transfection media was aspirated andreplaced with fresh HEK-T media and cells were incubated at 37° ° C./5%CO2. 24 hours after transfection, 500 μL samples of conditioned mediawere transferred to a separate plate and stored at −80° C., and theremaining media was aspirated. Cells were then washed once with ice-coldPBS and lysed in 250 μL/well ice-cold lysis buffer [25 mM Tris-HCl/12.5mM NaH2PO4/2 mM EGTA/1% Triton X-100/1× cOmplete Mini EDTA-free proteaseinhibitor]. Lysates were scraped, collected, clarified viacentrifugation at 10,000 g for 10′ at 4° C., and stored at −80° C. Thefollowing day, Clue activity in the conditioned media samples and Rlucactivity in the clarified lysates was measured using BioLux CyperidinaLuciferase (NEB #E3309) and Pierce Renilla Luciferase (Thermo Fisher#16167) assay kits, respectively, according to the manufacturer'sinstructions. Data in (FIG. 5, panel d) are the average of 10independent experiments.

The P46L mutation significantly reduced ATF4 activity, while nosignificant differences in activity were identified between Q22P and WTprotein (FIG. 5, panel d). Western blot analysis of lysates preparedfrom the HEK293T cells described above further revealed thatsteady-state levels of P46L ATF4 were reduced in comparison to WT ATF4control cDNA expressed under identical conditions (47.2% of WT) (FIG. 5,panels e, f). Data in (FIG. 5, panel e) are the average of 10independent experiments.

Levels of P46L ATF4 were also significantly more sensitive to treatmentwith the proteasome inhibitor, MG132, suggesting that the P46L mutationincreases its proteasomal degradation (FIG. 5, panel g). The datapresented in FIG. 5, panel g reveals greater contribution of proteasomaldegradation to P46L steady state levels as compared to WT ATF4. Cellsexpressing the FLAG-tagged ATF4 constructs indicated were treated withvehicle or MG132 (10 μM) for 2 hours, then lysed, blotted, andquantified. Data are the average of 8 independent experiments.Proteasomal degradation is a well-known, tightly regulated mechanismthat controls cellular levels of ATF4. (Andreev, D. E. et al., (2015)Elife 4, e03971; Waugh, J. L. & Sharma, N., (2013) Neurol. Clin. 31,969-86.) Therefore, this rare variant mutation may function bydysregulating this process.

Additional coding mutations identified in sporadic cervical dystoniacases (e.g. D35N, D35Y) showed significantly reduced activity,consistent with the direction of eIF2α pathway impairment that would bepredicted by the effects described in DYT1 here and DYT16 (FIG. 6).

This series of experiments provides evidence that deficient eIF2-alphasignaling may contribute not only to rare inherited dystonias, but alsoto sporadic forms of dystonia.

Example 9: DYT1 Patient Cells have Increased Basal Levels of a NegativeFeedback Regulator of the eIF2-Alpha Pathway

PRKRA and ATF4 are components of the eIF2-alpha pathway, providing amechanistic connection between eIF2-alpha signaling and dystonia.However, it remained unclear why eIF2-alpha signaling was disrupted inDYT1 (see Example 7; FIG. 5, panels a, b, supra). Because the ERstress-induced response of ATF4 translation was present but attenuatedin DYT1 patient cells, it was hypothesized that DYT1 is caused by anincrease in negative feedback mechanisms that attenuate54bosphor-eIF2-alpha signaling.

Two negative feedback proteins in the eIF2-alpha pathway are theeIF2-alpha phosphatase subunits, CreP and GADD34. A role for CreP wassuggested by our pharmacological studies in which only salubrinal (whichinhibits both CreP and GADD34) but not guanabenz (a specific inhibitorof GADD34) corrected delta E Torsin1a mislocalization. (See Example 4,supra.) We therefore measured levels of CreP under basal and ER-stressedconditions (FIG. 7). We determined at least two differences in CrePlevels between fibroblasts from DYT1 patients and healthy controls.First, basal CreP levels were significantly higher in fibroblasts fromDYT1 patients (FIG. 7, panels a, b), supporting our negative feedbackhypothesis. Second, whereas CreP levels in normal fibroblasts robustlyincreased after treatment with thapsigargin as expected (FIG. 6c ), CrePlevels in DYT1 patient fibroblasts were unresponsive to the treatment(FIG. 7, panel c). As the increase in CreP levels in response to stressis regulated by 55hosphor-eIF2-alpha-dependent translation (Marciniak,S. J. & Ron, D, (2006) Physiol. Rev. 86, 1133-49), the failure of CrePlevels to rise provides additional evidence that eIF2-alpha pathwaysignaling is deficient in DYT1.

These results support a mechanism in DYT1 dystonia whereby basallyincreased levels of negative feedback proteins impair acute,stress-responsive eIF2-alpha signaling (FIG. 7, panel d).

Example 10: Long Term Plasticity in the Cortico-Striatal Synapse byModulation of eIF2-Alpha Pathway

eIF2-alpha pathway dysfunction in a setting that does not involveexogenously expressed Torsin1a, mimicked the human genotype(heterozygosity), and involved the brain was examined. Dystonia is abrain disorder and although a disturbed ER stress response is aplausible mechanism for CNS disease, eIF2-alpha phosphorylation is alsoknown to have brain-specific roles in long-term synaptic plasticity(Costa-Mattioli and Sonenberg, 2006, Crit. Rev. Neurobiol. 18, 187-95;Di Prisco et al., 2014, Nat. Neurosci. 17, 1073-82; Trinh and Klann,2013, Neurobiol. Learn. Mem. 105, 93-9). Coincidentally, disruptedsynaptic plasticity in basal ganglia circuitry has long beenhypothesized as a disease mechanism for dystonia (reviewed in Petersonet al., 2010, Neurobiol. Dis. 37, 558-73). Furthermore, DYT1 mousemodels have disruptions of long-term synaptic plasticity in thestriatum—absence of type 5 metabotropic glutamate receptor(mGluRS)-dependent long-term synaptic depression (LTD) and increasedlong-term potentiation (LTP) magnitude (Martella et al., 2014,Neurobiol. Dis. 65, 124-32; Martella et al., 2009, Brain 132, 2336-49).In the hippocampus, eIF2-alpha phosphorylation is required formGluR-dependent LTD (Di Prisco et al., 2014, Nat. Neurosci. 17, 1073-82)and shifts that reduce the amount of phosphorylated protein have beenpreviously hypothesized to lower the threshold for LTP (Costa-Mattioliet al., 2007, Cell 129, 195-206). Thus, known brain-specific eIF2-alpharoles directly correlate with previously well-described alterations inDYT1 mouse model synaptic plasticity.

To directly test these predictions, whole-cell electrophysiology ofstriatal projection neurons was performed in acute brain slices toinduce long-term depression in the presence of eIF2-alphapathway-modulating drugs. Acute horizontal brain slices from the progenyof heterozygous AE Torsin1a knockin×homozygous Drd1a-tdTomato mice (ageP15-21) were prepared largely as described in (Trusel et al., 2015, CellRep. 13, 1353-65). LTD was induced as in Trusel et al., 2015, withmodifications.

Animals and Slice Preparation

Heterozygous delGAG Torsin1a knockin mice (courtesy of Dr. W. Dauer,University of Michigan) were crossed with homozygous Drd1a-tdTomato mice(Jackson Labs) and the progeny was used at postnatal day 15-21 forexperiments. Mice were anesthetized and intracardially perfused withhigh-sucrose solution (194 mM sucrose, 30 mM NaCl, 4.5 mM KCl, 2 mMMgCl2, 200 μM CaCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose,saturated with 95% 02 and 5% CO2). Animals were then decapitated, theirbrains dissected, and 300 μm horizontal slices were cut on a LeicaVTI200S vibratome. Slices were then transferred to artificialcerebrospinal fluid (ACSF; 124 mM NaCl, 2.5 mM KCl, 2 mM MgCl2, 2 mMCaCl2, 26 mM NaHCO3, 1.2 mM NaH2PO4, and 10 mM glucose, saturated with95% 02 and 5% CO2, pH 7.4, 300 mOsm/l) to equilibrate for at least 1 h.

Recording and Analysis

Single slices were transferred to a recording chamber and superfusedcontinuously with ACSF containing 50 μM picrotoxin at 32° C. and 3-4mL/min. Neurons were visualized using infrared differential interferencemicroscopy. Micropipettes were pulled (Narishige) from borosilicateglass tubes (King Precision Glass) for a final resistance of 2.5-4.5 MOwhen filled with internal solution (130 mM KSO4CH4, 5 mM KCl, 5 mM NaCl,100 μM EGTA, 10 mM HEPES, 2 mM MgCl2, 50 μM CaCl2, 2 mM ATP-Mg, 400 μMGTP-Na, pH 7.3, 290 mOsm/l). Evoked excitatory postsynaptic potentials(EPSPs) were recorded in the dorsolateral striatum, while stimulatingevery 30 seconds with a concentric bipolar electrode (FHC) in corticallayer V (see Trusel et al., Cell Reports, 2015). To induce long-termdepression, 4 trains of 100 Hz stimulation (every 10 s) were appliedwhile the postsynaptic cell was depolarized to −50 mV. Baseline EPSPswere recorded for at least 10 min or until a stable baseline wasreached. Data were acquired by pClamp v10 and analyzed using Clampfitv10.4, Origin v8.0, and GraphPad Prism v6.

Results

eIF2-alpha signaling was assessed to determine if it normally plays arole in striatal mGluRS LTD. Pre-incubation with ISRIB (5 nM), a blockerof eIF2-alpha pathway signaling, robustly inhibited LTD in WT brainslices (FIG. 8, panel A). Additionally, it was notable that whilecontrol vehicle-treated slices display a range of responses, none weregreater than the baseline amplitude; however, in the presence of ISRIB,potentiating responses were now observed.

To test whether augmenting eIF2-alpha signaling in DYT1 model mice wouldnormalize synaptic plasticity, brain slices from mice heterozygous forthe DYT1 knockin mutation (delGAG) were treated with 20 μM Sal-003, aninhibitor of eIF2α phosphatases. In comparison to vehicle-treated brainslices, Sal-003 restored long-term depression (FIG. 8, panel B) and tolevels that once again approximate the normal magnitude (FIG. 8, panel Band C). These observations demonstrate that eIF2-alpha signalingnormally plays a role in brain processes that are disrupted in DYT1model mice and that acute enhancement of eIF2-alpha phosphorylation issufficient to restore normal brain plasticity to disease model mice.

Example 11: Anti-Retroviral “Avir” Drugs are Useful for TreatingDystonia, and Dystonia Associated Disorders

The HIV protease inhibitors ritonavir, lopinavir, saquinovir,nelfinavir, and indinavir corrected TorsinA localization in adose-dependent fashion, with no cellular toxicity observed at effectivedoses (Table 5, FIG. 10). Furthermore, in orthogonal assays, ritonavir,lopinavir, and saquinovir corrected secretory deficits (Table 5), aphenotype also present in Dyti patient-derived fibroblasts. (Cao, S. etal. (2010) Dis. Model. Mech. 3, 386-96.) Subsequent testing ofadditional HIV protease inhibitors supported the activity of this classof compounds and generated favorable structure-activity relationship(SAR) data from which to design improved compounds (Tables 5 and 6). Theresults with a subset of compounds were also observed in a Luciferasesecretion assay (Tables 5 and 6).

TABLE 5 Delta-Torsin1a mislocalization assay and C.Luc. SectretionAssays ΔE TorsinA Collected Mislocalization Assay C.Luc. Secretion AssayActivity Localization Localization Cytotoxicity Secretion Secretion DataEC50 (μM) Max Efficacy EC50 (μM) EC50 (μM) Max Efficacy Ritonavir 4.1187% None 3.72 49% Lopinavir 4.69 70% 37.2 0.98 177%  Saquinavir 9.41 88%23.6 8.09 34% Nelfinavir 3.49 80% 6.05 No Activity Indinavir 26.5 114% 35.2 No Activity Atazanavir No Activity None No Activity Amprenavir NoActivity None No Activity Darunavir No Activity None No Activity

Table 6 presents additional data showing that the indicated HIV proteaseinhibitors correct DYT1 phenotypes. % DYT1 Pathology is according tomethods for high-throughput assay screening analysis described above, inwhich % cells with punctate EGFP signal following EGFP-Torsin1a proteinexpression are measured as “% selected cells.” Efficacy and Max Responserefer to activity on a normalized scale in which the % selected cells inthe cell line expressing WT-TorsinA is set to 0% and the %/selectedcells in the cell line expressing DYT1 Mutant Torsin1a is set to 100%pathology. Efficacy is calculated as the magnitude of the pathologycorrection relative to the mutant Torsin1a pathology. For example anefficacy=−100% would be equivalent to the WT TorsinA phenotype, and 0%would be equivalent to the Mutant TorsinA phenotype. CCv2 refers to thedose response Curve Class annotation, and AC refers to ActiveConcentration. (See Inglese, J., et al. (2006), PNAS, 103:11473-11478.)

TABLE 6 HIV protease inhibitors correct DYT1 phenotypes % pathologyCytotox AC50 Max AC50 Max Sample Name CC-v2 (μM) Efficacy Resp CC-v2(μm) Efficacy Resp Viracept −2.3 5.26 −119.39 −94.06 −1.1 10.49 −106.32−97.72 Saquinavir −1.1 8.33 −85.98 −84.62 −2.1 20.93 −105.74 −96.01mesylate Indinavir −2.1 29.57 −114.60 −97.76 −3 37.22 −80.64 −67.80sulfate Amprenavir 4 4 Ritonavir −1.1 2.96 −92.96 −82.60 4 Lopinavir−1.3 4.69 −69.97 −89.03 −3 37.22 −69.87 −55.19 Darunavir 4 4 Atazanavir4 4 Nelfinavir −1.1 2.70 −75.26 −92.46 −1.1 6.05 −98.05 −95.64 mesylaehydrate Des-hydroxy −3 8.33 −82.44 −33.96 −2.2 20.93 −77.66 −66.67Lopinavir

Western blot analyses were conducted to determine the effect ofLopinavir on ATF4, CreP, pEIF2-alpha, and β-actin abundance in EGFP-ΔETorsin1a expression assay cell lines. The HIV protease inhibitorLopinavir increases phosphorylation of EIF2-alpha and ATF4 abundance ina dose dependent manner through down-regulation of steady state levelsof the EIF2-alpha protein phosphatase 1 regulatory subunit, CreP, asdetermined in the EGFP-ΔE Torsin1a expression assay cell line (FIG. 11).This down regulation of CreP is in keeping with the HIV proteaseinhibitor mediated CreP regulation. (See De Gassart et al. PNAS 2015.)

1. A method of treating a subject having dystonia comprisingadministering one or more of an HIV aspartyl protease inhibitor.
 2. Themethod of claim 1, wherein the HIV aspartyl protease inhibitor is acompound of formula:

or pharmaceutically acceptable salts thereof, wherein each n isindependently selected from 0, 1, 2, 3, and 4; R¹ is hydrogen or C₁-C₆alkyl; R² and R³ are each independently selected from halogen, hydroxy,C₁-C₆ alkyl, and C₁-C₆ alkoxy; R⁴ is —C(O)R⁸, —C(O)OR⁸, —C(O)NHR⁸, or—C(O)N(C₁-C₆ alkyl)R⁸, where R⁸ is C₁-C₆ alkyl, aryl, heteroaryl,heterocyclyl, arylC₁-C₆ alkyl-, heteroarylC₁-C₆ alkyl-, orheterocyclylC₁-C₆ alkyl-, each optionally substituted with one or moregroups selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino; R⁵ is selectedfrom hydrogen and C₁-C₆ alkyl, or R⁵ and R⁸ together with atoms to whichthey are attached form a heterocyclyl optionally substituted with one ormore of groups selected from halogen, cyano, nitro, C₁-C₆ alkyl,hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo,and thio; and R⁶ is —Z—R⁷, wherein Z is absent, —C₁-C₆ alkylene-,—O—C₁-C₆ alkylene-, —C₁-C₆ alkylene-O—, —NH—C₁-C₆ alkylene-, —C₁-C₆alkylene-NH—, —O—, —NH—, or —N(C₁-C₆ alkyl)-; and R⁷ is aryl,heteroaryl, or heterocyclyl, each optionally substituted with one ormore groups selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy,C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino.
 3. Themethod of claim 2, wherein the compound is of formula:


4. The method of claim 2, wherein each n is
 0. 5. The method of claim 2,wherein R¹ is C₁-C₆ alkyl.
 6. The method of claim 2, wherein R⁴ is—C(O)R⁸, —C(O)OR⁸, —C(O)NHR⁸, or —C(O)N(C₁-C₆ alkyl)R⁸, where R⁸ isC₁-C₆ alkyl, aryl, heteroaryl, heterocyclyl, arylC₁-C₆ alkyl-,heteroarylC₁-C₆ alkyl-, or heterocyclylC₁-C₆ alkyl-, each optionallysubstituted with one or more of groups selected from halogen, cyano,nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, anddiC₁-C₆ alkylamino; and R⁵ is selected from hydrogen and C₁-C₆ alkyl. 7.The method of claim 6, wherein R⁴ is —C(O)NHR⁸ or —C(O)N(C₁-C₆ alkyl)R⁸,where R⁸ is arylC₁-C₆ alkyl-, heteroarylC₁-C₆ alkyl-, orheterocyclylC₁-C₆ alkyl-, each optionally substituted with one or moreof groups selected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy,C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino; and R⁵ ishydrogen.
 8. The method of claim 2, wherein —N(—R⁴)(—R⁵) is of formula:


9. The method of claim 2, wherein R⁵ and R⁸ together with atoms to whichthey are attached form a heterocyclyl optionally substituted with one ormore of groups selected from halogen, cyano, nitro, C₁-C₆ alkyl,hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, diC₁-C₆ alkylamino, oxo,and thio.
 10. The method of claim 9, wherein —N(—R⁴)(—R⁵) is of formula:


11. The method of claim 2, wherein Z is —C₁-C₆ alkylene-, —O—C₁-C₆alkylene-, or —C₁-C₆ alkylene-O—; and R⁷ is aryl, heteroaryl, orheterocyclyl, each optionally substituted with one or more of groupsselected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy,amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino.
 12. The method of claim2, wherein Z is —O-methylene- or -methylene-O—; and R⁷ is aryl orheteroaryl, each optionally substituted with one or more of groupsselected from halogen, cyano, nitro, C₁-C₆ alkyl, hydroxy, C₁-C₆ alkoxy,amino, C₁-C₆ alkylamino, and diC₁-C₆ alkylamino.
 13. The method of claim2, wherein R⁶ is of formula:


14. The method of claim 1, wherein the HIV aspartyl protease inhibitoris selected from ritonavir, liponavir, saquinavir, nelfinavir,indinavir, and a combination thereof.
 15. The method of claim 1, whereinthe HIV aspartyl protease inhibitor is selected from ritonavir,liponavir, saquinavir, and a combination thereof.